Method of synthesis and testing of cominatorial libraries using microcapsules

ABSTRACT

Methods for use in the synthesis and identification of molecules which bind to a target component of a biological system or modulate the activity of a target are described.

This application is a continuation of U.S. Ser. No. 11/238,257, filedSep. 29, 2005, allowed, which is a continuation of PCT/GB2004/001352,filed Mar. 31, 2004, which claims priority to United Kingdom applicationGB0307428.3, filed Mar. 31, 2003, the entire contents of each of whichare incorporated herein by reference.

The present invention relates to methods for use in the synthesis andidentification of molecules which bind to a target component of abiochemical system or modulate the activity of a target.

Over the past decade, high-throughput screening (HTS) of compoundlibraries has become a cornerstone technology of pharmaceuticalresearch. Investment into HTS is substantial. A current estimate is thatbiological screening and preclinical pharmacological testing aloneaccount for ˜14% of the total research and development (R&D)expenditures of the pharmaceutical industry (Handen, Summer 2002). HTShas seen significant improvements in recent years, driven by a need toreduce operating costs and increase the number of compounds and targetsthat can be screened. Conventional 96-well plates have now largely beenreplaced by 384-well, 1536-well and even 3456-well formats. This,combined with commercially available plate-handling robotics allows thescreening of 100,000 assays per day, or more, and significantly cutscosts per assay due to the miniaturisation of the assays.

HTS is complemented by several other developments. Combinatorialchemistry is a potent technology for creating large numbers ofstructurally related compounds for HTS. Currently, combinatorialsynthesis mostly involves spatially resolved parallel synthesis. Thenumber of compounds that can be synthesised is limited to hundreds orthousands but the compounds can be synthesised on a scale of milligramsor tens of milligrams, enabling full characterisation and evenpurification. Larger libraries can be synthesised using split synthesison beads to generate one-bead-one compound libraries. This method ismuch less widely adopted due to a series of limitations including: theneed for solid phase synthesis; difficulties characterising the finalproducts (due to the shear numbers and small scale); the small amountsof compound on a bead being only sufficient for one or a few assays; thedifficulty in identifying the structure of a hit compound, which oftenrelies on tagging or encoding methods and complicates both synthesis andanalysis. Despite this split synthesis and single bead analysis stillhas promise. Recently there have been significant developments inminiaturised screening and single bead analysis. For example, printingtechniques allow protein-binding assays to be performed on a slidecontaining 10,800 compound spots, each of 1 nl volume (Hergenrother etal., 2000). Combichem has so far, however, generated only a limitednumber of lead compounds. As of April 2000, only 10 compounds with acombinatorial chemistry history had entered clinical development and allbut three of these are (oligo)nucleotides or peptides (Adang andHermkens, 2001). Indeed, despite enormous investments in both HTS andcombinatorial chemistry during the past decade the number of new drugsintroduced per year has remained constant at best.

Dynamic combinatorial chemistry (DCC) can also be used to create dynamiccombinatorial libraries (DCLs) from a set of reversibly interchangingcomponents, however the sizes of libraries created and screened to dateare still fairly limited (≦40,000) (Ramstrom and Lehn, 2002).

Virtual screening (VS) (Lyne, 2002), in which large compound bases aresearched using computational approaches to identify a subset ofcandidate molecules for testing may also be very useful when integratedwith HTS. However, there are to date few studies that directly comparethe performance of VS and HTS, and further validation is required.

Despite all these developments, current screening throughput is stillfar from adequate. Recent estimates of the number of individual genes inthe human genome (˜30,000) and the number of unique chemical structurestheoretically attainable using existing chemistries suggests that anenormous number of assays would be required to completely map thestructure-activity space for all potential therapeutic targets (Burbaum,1998).

Hence, a method with the capability to both create and screen vastnumbers (≧10¹⁰) of compounds quickly, using reaction volumes of only afew femtolitres, and at very low cost should be of enormous utility inthe generation of novel drug leads.

SUMMARY OF THE INVENTION

The invention, in a first aspect, provides a method for preparing arepertoire of compounds comprising the steps of:

(a) compartmentalising two or more sets of primary compounds intomicrocapsules; such that a proportion of the microcapsules containsmultiple copies of one or more compounds representative of each of saidsets, and wherein said one or more compounds represents a subset of theset of primary compounds; and

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets.

A compound is “representative” of a set where is member of said set;advantageously, therefore, each microcapsule contains compound(s) fromeach set. Although a microcapsule may contain more than one differentcompound from each set, it contains only a proportion of said set—i.e. asubset. The subset of a set advantageously represents no more that 10%of the members of the set; preferably, this figure is 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1% or less.

Most advantageously a microcapsule contains only a single compound fromeach set of primary compounds.

The sets of primary compounds used in the method of the invention canconsist of any number of different compounds. At least a first setcomprises two or more compounds; but the other set may be a singlecompound. Preferably, if a first set is a single compound, at least onefurther set comprises a repertoire of compounds. The larger thisrepertoire, the greater the number of different secondary compounds thatwill be generated.

Preferably, at least one set of compounds comprises a repertoire ofdifferent compounds. At least one set, however, may consist of a singlecompound, such that secondary compounds are all constructed based on orcontaining the single compound used in one set. The greater the numberof sets, and the greater the diversity of each set, the greater thefinal diversity of the secondary compounds generated.

Advantageously, in step (a) the number of different compounds percompartment will be equivalent to the number of primary compoundsforming the secondary compound in step (b).

In a second aspect, the invention provides a method for identifyingprimary compounds which react together to form secondary compoundscapable of binding to or modulating the activity of a target, comprisingthe steps of:

(a) compartmentalising two or more sets of primary compounds intomicrocapsules; such that a proportion of the microcapsules contains twoor more compounds;

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget.

In a third aspect, the invention provides a method for synthesisingcompounds with enhanced ability to bind to or modulate the activity ofthe target, comprising the steps of:

(a) compartmentalising into microcapsules subsets of primary compoundsidentified in step (c) of the second aspect of the invention and,optionally, compartmentalising additional sets of primary compounds;

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget.

Advantageously, steps (a) to (c) can be repeated, but after the firstcycle, step (a) comprises compartmentalising subsets of primarycompounds identified in step (c) into microcapsules and, optionally,compartmentalising additional sets of compounds.

In a fourth aspect, the invention provides a method for identifyingindividual compounds which bind to or modulate the activity of thetarget, comprising the steps of:

(a) compartmentalising into microcapsules a primary compound identifiedin step (c) of the second or third aspect of the invention andadditional sets of primary compounds;

(b) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(c) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget.

If the secondary compound is formed by the chemical reaction betweenmore than two primary compounds it can be identified by iterativelyrepeating steps (a) to (c), but after the first cycle, step (c)comprises compartmentalising the primary compound identified in step (c)of the second or third aspect of the invention, a primary compoundidentified in step (c) of each of the previous cycles (of the fourthaspect of the invention) and additional sets of primary molecules.

Preferably, the desired activity is selected from the group consistingof a binding activity and the modulation of the activity of a target.Advantageously, the target is compartmentalised into microcapsulestogether with the compounds.

Sets of compounds may be compartmentalised in different ways to achieveencapsulation of multiple copies of two or more compounds intomicrocapsules.

For example, small aliquots of an aqueous solution of each compound canbe deposited into an oil phase (advantageously containing surfactantsand/or other stabilising molecules) whilst applying mechanical energy,thereby dispersing each compound into multiple aqueous microcapsules,each of which contains (for the most part) a single sort of compound butmultiple copies thereof. Advantageously, the compounds can be depositedinto the oil phase in the form of droplets generated using inkjetprinting technology (Calvert, 2001; de Gans et al., 2004), and moreadvantageously by piezoelectric drop-on-demand (DOD) inkjet printingtechnology. Inkjet printing technology can be used to mix primarycompounds and, optionally, other reagents (e.g the target and reagentsto assay target activity) immediately prior to forming the emulsion.Advantageously, multiple compounds can be mixed with multiple targets ina combinatorial manner.

Thus, step (a) above can be modified such that it comprises formingseparate emulsion compartments containing two or more compounds andmixing the emulsion compartments to form an emulsified compoundrepertoire wherein a subset of the repertoire is represented in multiplecopies in any one microcapsule.

Moreover, compound libraries can be compartmentalised in highlymonodisperse microcapsules produced using microfluidic techniques. Forexample, aliquots of each compound can be compartmentalised into one ormore aqueous microcapsules (with less than 3% polydispersity) inwater-in-oil emulsions created by droplet break off in a co-flowingsteam of oil (Umbanhowar et al., 2000). Advantageously, the aqueousmicrocapsules are then transported by laminar-flow in a stream of oil inmicrofluidic channels (Thorsen et al., 2001). The microcapsulescontaining single compounds can, optionally, be split into two or moresmaller microcapsules using microfluidics (Link et al., 2004; Song etal., 2003). Microcapsules containing primary compounds can be fused withother microcapsules (Song et al., 2003) to form secondary compounds.Microcapsules containing compounds can also, optionally, be fused withmicrocapsules containing a target. A single microcapsule containing atarget can, optionally, be split into two or more smaller microcapsuleswhich can subsequently be fused with microcapsules containing differentcompounds, or compounds at different concentrations. Advantageously, acompound and a target can be mixed by microcapsule fusion prior to asecond microcapsule fusion which delivers the reagents necessary toassay the activity of the target (e.g. the substrate for the target ifthe target is an enzyme). This allows time for the compound to bind tothe target. The microcapsules can be analysed and, optionally, sortedusing microfluidic devices (Fu et al., 2002).

In a further aspect, there is provided a method for preparing arepertoire of compounds comprising the steps of:

(a) attaching two or more sets of primary compounds onto microbeads;

(b) compartmentalising the microbeads into microcapsules such that aproportion of the microcapsules contains two or more microbeads;

(c) releasing at least one of the sets of primary compounds from themicrobeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets.

Advantageously, the compounds are cleavable from the beads. Where morethan two sets of compounds are used, all the sets with the exception ofone are cleavable; preferably, they are all cleavable. The compounds maybe attached to the microbeads by photochemically cleavable linkers.

In a still further aspect, the invention provides a method foridentifying primary compounds which react together to form secondarycompounds capable of binding to or modulating the activity of a target,comprising the steps of

(a) attaching two or more sets of primary compounds onto microbeads;

(b) compartmentalising the microbeads into microcapsules together withthe target such that many compartments contain two or more microbeads;

(c) releasing the primary compounds from the microbeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(e) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget.

Advantageously, in step (b) the modal number of microbeads percompartment will be equivalent to the number of primary compoundsforming the secondary compound in step (d).

In a further aspect, the invention provides a method for synthesisingcompounds with enhanced ability to bind to or modulate the activity ofthe target, comprising the steps of:

(a) attaching onto microbeads subsets of primary compounds identified instep (e) of the second aspect of the invention and, optionally,attaching additional sets of primary compounds;

(b) compartmentalising the microbeads into microcapsules together withthe target such that many compartments contain two or more microbeads;

(c) releasing the primary compounds from the microbeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(e) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget.

Advantageously, steps (a) to (e) can be repeated, but after the firstcycle, step (a) comprises attaching onto microbeads subsets of primarycompounds identified in step (e) and, optionally, attaching additionalsets of compounds.

In a further aspect, the invention provides a method for identifyingindividual compounds which bind to or modulate the activity of thetarget, comprising the steps of:

(a) attaching onto microbeads a primary compound identified in step (e)of the second or third aspect of the invention and additional sets ofprimary compounds;

(b) compartmentalising the microbeads into microcapsules together withthe target such that many compartments contain two or more microbeads;

(c) releasing the primary compounds from the microbeads;

(d) forming secondary compounds in the microcapsules by chemicalreactions between primary compounds from different sets; and

(e) identifying subsets of primary compounds which react to formsecondary compounds which bind to or modulate the activity of thetarget;

If the secondary compound is formed by the chemical reaction betweenmore than two primary compounds it can be identified by iterativelyrepeating steps (a) to (e), but after the first cycle, step (a)comprises attaching onto microbeads the primary compound identified instep (e) of the second or third aspect of the invention, a primarycompound identified in step (e) of each of the previous cycles (of thefourth aspect of the invention) and additional sets of primarymolecules.

Preferably, the desired activity is selected from the group consistingof a binding activity and the modulation of the activity of a target.Advantageously, the target is compartmentalised into microcapsulestogether with the microbeads.

According to a preferred implementation of the present invention, thecompounds may be screened according to an activity of the compound orderivative thereof which makes the microcapsule detectable as a whole.Accordingly, the invention provides a method wherein a compound with thedesired activity induces a change in the microcapsule, or a modificationof one or more molecules within the microcapsule, which enables themicrocapsule containing the compound and, optionally, the microbeadcarrying it to be identified. In this embodiment, therefore, themicrocapsules are either: (a) physically sorted from each otheraccording to the activity of the compound(s) contained therein, and thecontents of the sorted microcapsules analysed to determine the identityof the compound(s) which they contain; or (b) analysed directly withoutsorting to determine the identity of the compound(s) which themicrocapsules contain.

According to a preferred embodiment of the present invention, thescreening of compounds may be performed by, for example:

(I) In a first embodiment, the microcapsules are screened according toan activity of the compound or derivative thereof which makes themicrocapsule detectable as a whole. Accordingly, the invention providesa method wherein a compound with the desired activity induces a changein the microcapsule, or a modification of one or more molecules withinthe microcapsule, which enables the microcapsule containing the compoundand the microbead carrying it to be identified. In this embodiment,therefore, the microcapsules are either: (a) physically sorted from eachother according to the activity of the compound(s) contained therein,the contents of the sorted microcapsules optionally pooled into one ormore common compartments, and the microcapsule contents analysed todetermine the identity of the compound(s); or (b) analysed directlywithout sorting to determine the identity of the compound(s) which themicrocapsules contained. Where the microcapsule contains microbeads, themicrobeads can be analysed to determine the compounds with which theyare coated.

(II) In a second embodiment, microbeads are analysed following poolingof the microcapsules into one or more common compartments. In thisembodiment, a compound having the desired activity modifies themicrobead which carried it (and which resides in the same microcapsule)in such a way as to make it identifiable in a subsequent step. Thereactions are stopped and the microcapsules are then broken so that allthe contents of the individual microcapsules are pooled. Modifiedmicrobeads are identified and either: (a) physically sorted from eachother according to the activity of the compound(s) coated on themicrobeads, and the sorted microbeads analysed to determine the identityof the compound(s) with which they are/were coated; or (b) analyseddirectly without sorting to determine the identity of the compound(s)with which the microbeads are/were coated. It is to be understood, ofcourse, that modification of the microbead may be direct, in that it iscaused by the direct action of the compound, or indirect, in which aseries of reactions, one or more of which involve the compound havingthe desired activity, leads to modification of the microbead.Advantageously, the target is bound to the microbead and is a ligand andthe compound within the microcapsule binds, directly or indirectly, tosaid ligand to enable the isolation of the microbead. In anotherconfiguration, a substrate for the target is and is bound to themicrobead, and the activity of the compound within the microcapsuleresults, directly or indirectly, in the conversion of said substrateinto a product which remains part of the microbead and enables itsisolation. Alternatively, the activity of the compound may prevent orinhibit the conversion of said substrate into product. Moreover, theproduct of the activity of the compound within the microcapsule canresult, directly or indirectly, in the generation of a product which issubsequently complexed with the microbead and enables itsidentification.

(III) In a third embodiment, the microbeads are analysed followingpooling of the microcapsules into one or more common compartments. Inthis embodiment, a compound with a desired activity induces a change inthe microcapsule containing the compound and the microbead which carriesit. This change, when detected, triggers the modification of themicrobead within the compartment. The reactions are stopped and themicrocapsules are then broken so that all the contents of the individualmicrocapsules are pooled. Modified microbeads are identified and either:(a) physically sorted from each other according to the activity of thecompound(s) coated on the microbeads, and the sorted microbeads analysedto determine the identity of the compound(s) with which they are/werecoated; or (b) analysed directly without sorting to determine theidentity of the compound(s) with which the microbeads are/were coated.

The microcapsules or microbeads may be modified by the action of thecompound(s) such as to change their optical properties. For example, themodification of the microbead can enable it to be further modifiedoutside the microcapsule so as to induce a change in its opticalproperties.

In another embodiment, the change in optical properties of themicrocapsules or microbeads is due to binding of a compound withdistinctive optical properties to the target.

Moreover, the change in optical properties of the microcapsules ormicrobeads can be due to binding of a target with distinctive opticalproperties by the compound.

The change in the optical properties of the micro capsule may be due tomodulation of the activity of the target by the compound. The compoundmay activate or inhibit the activity of the target. For example, if thetarget is an enzyme, the substrate and the product of the reactioncatalysed by the target can have different optical properties.Advantageously, the substrate and product have different fluorescenceproperties. In the case where the microcapsules contain microbeads, boththe substrate and the product can have similar optical properties, butonly the product of the reaction, and not the substrate, binds to, orreacts with, the microbead, thereby changing the optical properties ofthe microbead.

The change in optical properties of the microcapsules or microbeads canalso be due to the different optical properties of the target and theproduct of the reaction being selected. Where both target and producthave similar optical properties, only the product of the reaction beingselected, and not the target, binds to, or reacts with, the microbead,thereby changing the optical properties of the microcapsules ormicrobeads.

In a further configuration, further reagents specifically bind to, orspecifically react with, the product (and not the substrate) attached toor contained in the microcapsule or microbead, thereby altering theoptical properties of the microcapsule or microbead

Advantageously, microcapsules or microbeads are modified directly orindirectly by the activity of the compound are further modified byTyramide Signal Amplification (TSA™; NEN), resulting directly orindirectly in a change in the optical properties of said microcapsulesor microbeads thereby enabling their separation.

It is to be understood that the detected change in the compartment maybe caused by the direct action of the compound, or indirect action, inwhich a series of reactions, one or more of which involve the compoundhaving the desired activity leads to the detected change.

Where the compounds are attached to beads, the density with whichcompounds are coated onto the microbeads, combined with the size of themicrocapsule will determine the concentration of the compound in themicro capsule. High compound coating densities and small microcapsuleswill both give higher compound concentrations which may be advantageousfor the selection of molecules with a low affinity for the target.Conversely, low compound coating densities and large microcapsules willboth give lower compound concentrations which may be advantageous forthe selection of molecules with a high affinity for the target.

Preferably, microencapsulation is achieved by forming an emulsion.

The microbead can be nonmagnetic, magnetic or paramagnetic.

Advantageously, the microcapsules or microbeads are analysed bydetection of a change in their fluorescence. For example, microbeads canbe analysed by flow cytometry and, optionally sorted using afluorescence activated cell sorter (FACS). The different fluorescenceproperties of the target and the product can be due to fluorescenceresonance energy transfer (FRET).

In a further embodiment, the internal environment of the microcapsulescan be modified by the addition of one or more reagents to thecontinuous phase of the emulsion. This allows reagents to be diffused into the microcapsules during the reaction, if necessary.

The invention moreover relates to a method according to the precedingaspects, further comprising the step of isolating the secondary compoundproduced by reaction of the primary compounds and optionally furthercomprising the step of manufacturing one or more secondary compounds.

The invention also provides for a product when identified according tothe invention. As used in this context, a “product” may refer to anycompound, selectable according to the invention.

Further embodiments of the invention are described in the detaileddescription below and in the accompanying claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Examples of PTP1B inhibitors. Compounds with abis-difluoromethylene phosphonate moiety (e.g. 2) have significantlymore potency than those with a single moiety (e.g. 1).

FIG. 2. Screening PTP1B inhibitors using IVC. Polystyrene beads withsurface carboxylate groups, died with orange or red fluorochromes(Fulton et al., 1997), are derivatised with a phosphopeptide PTP1Bsubstrate, and either PTP1B inhibitors or non-inhibitory compounds,attached via a cleavable linker (1). After mixing the beads, singlebeads and target enzyme (PTP1B) are colocalised in a microcompartment byforming a water-in-oil emulsion (2). The compound is released(photochemically) (3). Inhibitors reduce the amount of substrateconverted to product (dephosphorylated peptide) (4). The enzyme reactionis stopped and the emulsion is broken (5). After labelling with greenfluorescent anti-substrate antibodies, beads are analysed by 3-colourflow cytometry to simultaneously determine extent of inhibition and thecompound on the beads (6). Compound libraries can be coupled tooptically tagged beads (see below) and rapidly decoded by flow cytometry(at up to 100,000 beads s⁻¹). Hit compounds are re-synthesised forfurther characterisation (7) or elaborated and rescreened in a processof synthetic evolution (8).

FIG. 3. Synthesising PTP1B inhibitors in an emulsion. Two types of beadsare created, differentially labelled with orange and red fluorochromes,and derivatised with two types of molecule, A or B (neither, one, orboth of which contain a difluoromethylene phosphonate moiety), attachedvia a reversible connection (a Schiff base). Beads are emulsified togive, on average, two beads per compartment. The molecules, A & B, arereleased from the beads in the compartment and react to form a newmolecule, A-B, (in solution). If A-B is a PTP1B inhibitor the PTP1Bsubstrate also on the beads is not dephosphorylated and these beadsidentified by flow cytometry as FIG. 2.

FIG. 4. Small molecule evolution using four-component reactions. Foursets of 25 beads are created, each derivatised with one of 25 variantsof molecules A, B, C or D, emulsified to give, on average, 4 beads percompartment, the compounds released to synthesise a large combinatorialrepertoire (4×10⁵) in situ and screened as FIG. 2. Low affinityinhibitors will be ‘recombined’ by re-screening mixtures of beadscarrying moieties identified in inhibitors. Beads carrying a moietyfound in inhibitors (e.g. A₁₀) can also be mixed with complete sets ofbeads coated with B, C and D and screened. If a moiety (say B₈) is thenidentified as a component of an inhibitor, beads coated with A₁₀ and B₈can be mixed with complete sets of beads C and D and the processrepeated. This process of ‘mutation’ also results in deconvolution.After fixing three of the four moieties in active compounds,deconvolution can be completed using multiplex bead analysis as above.Compounds can be re-diversified or ‘mutated’ using bead sets carryingvariant, exploded sets of the molecules used in the original libraries.

FIG. 5. Compartmentalisation of small molecules in water-in-fluorocarbonemulsions. Water-in-perfluorooctyl bromide emulsions were madecontaining texas red (1 mM) and calcein (1 mM) in the aqueous phase byhomogenisation as described in example 6 The two emulsions were mixed byvortexing and imaged by epifluorescence microscopy after 24 hours. Noexchange of texas-red (red fluorescence) and calcein (greenfluorescence) between microdroplets could be observed.

FIG. 6. Primary compounds for the synthesis of PTP1B inhibitors. Anamine (A) and an aldehyde (B) with difluoromethylene phosphonatemoieties. Amine A reacts with aldehyde B in the aqueous microcapsules ofa water-in-oil emulsion to generate the imine C which is a potent PTP1Binhibitor. C can be reduced in situ using cyanoborohydride to generatethe stable amine D.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “microcapsule” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.In essence, however, a microcapsule is an artificial compartment whosedelimiting borders restrict the exchange of the components of themolecular mechanisms described herein which allow the identification ofthe molecule with the desired activity. The delimiting borderspreferably completely enclose the contents of the microcapsule.Preferably, the microcapsules used in the method of the presentinvention will be capable of being produced in very large numbers, andthereby to compartmentalise a library of compounds. Optionally, thecompounds can be attached to microbeads. The microcapsules used hereinallow mixing and sorting to be performed thereon, in order to facilitatethe high throughput potential of the methods of the invention. Arrays ofliquid droplets on solid surfaces, and multiwell plates, are notmicrocapsules as defined herein.

A “proportion” of the microcapsules, which is defined as comprising twoor more compounds, or two or microbeads, is any fraction of themicrocapsules in question, including all of said microcapsules.Advantageously, it is at least 25% thereof, preferably 50%, and morepreferably 60%, 70%, 80%, 90% or 95%.

The term “microbead” is used herein in accordance with the meaningnormally assigned thereto in the art and further described hereinbelow.Microbeads, are also known by those skilled in the art as microspheres,latex particles, beads, or minibeads, are available in diameters from 20nm to 1 mm and can be made from a variety of materials including silicaand a variety of polymers, copolymers and terpolymers. Highly uniformderivatised and non-derivatised nonmagnetic and paramagneticmicroparticles (beads) are commercially available from many sources(e.g. Sigma, Bangs Laboratories, Luminex and Molecular Probes) (Fornusekand Vetvicka, 1986).

Microbeads can be “compartmentalised” in accordance with the presentinvention by distribution into microcapsules. For example, in apreferred aspect the microbeads can be placed in a water/oil mixture andemulsified to form a water-in-oil emulsion comprising microcapsulesaccording to the invention. The concentration of the microbeads can beadjusted to control the number of microbeads, which on average, appearin each microcapsule.

The term “compound” is used herein in accordance with the meaningnormally assigned thereto in the art. The term compound is used in itsbroadest sense i.e. a substance comprising two or more elements in fixedproportions, including molecules and supramolecular complexes. Thisdefinition includes small molecules (typically <500 Daltons) which makeup the majority of pharmaceuticals. However, the definition alsoincludes larger molecules, including polymers, for example polypeptides,nucleic acids and carbohydrates, and supramolecular complexes thereof.

The term “primary compound” is used herein to indicate a compound whichis compartmentalised in a microcapsule or coupled to a bead.

The term “secondary compound” is used herein to indicate a compoundwhich is formed by the reaction between two or more primary compounds ina microcapsule, optionally after the release of at least one of theprimary molecules from a microbead. Advantageously, all primarymolecules are released from the microbeads. The secondary compound maybe the result of a covalent or non-covalent reaction between the primarycompounds.

The term “scaffold” is used herein in accordance with the meaningnormally assigned thereto in the art. That is to say a core portion of amolecule common to all members of a combinatorial library (Maclean etal., 1999). Secondary compounds may optionally comprise scaffolds.

A “repertoire” of compounds is a group of diverse compounds, which mayalso be referred to as a library of compounds. Repertoires of compoundsmay be generated by any means known in the art, including combinatorialchemistry, compound evolution, or purchased from commercial sources suchas Sigma Aldrich, Discovery Partners International, Maybridge andTripos. A repertoire advantageously comprises at least 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or more different compounds, whichmay be related or unrelated in structure or function.

A “set” of compounds may be a repertoire of compounds or any part of arepertoire, including a single compound species. The invention envisagesthe use of two or more sets of compounds, which are reacted together.The sets may be derived from a single repertoire, or a plurality ofdifferent repertoires.

Compounds can be “released” from a microbead by cleavage of a linkerwhich effects the attachment of the compound to the microbead. Releaseof the compounds from the microbead allows the compounds to interactmore freely with other contents of the microcapsule, and to be involvedin reactions therein and optionally to become combined with otherreagents to form new compounds, complexes, molecules or supramolecularcomplexes. Cleavage of linkers can be performed by any means, with meanssuch as photochemical cleavage which can be effected from without themicrocapsule being preferred. Photochemically cleavable linkers areknown in the art (see for example (Gordon and Balasubramanian, 1999))and further described below.

As used herein, the “target” is any compound, molecule, orsupramolecular complex. Typical targets include targets of medicalsignificance, including drug targets such as receptors, for example Gprotein coupled receptors and hormone receptors; transcription factors,protein kinases and phosphatases involved in signalling pathways; geneproducts specific to microorganisms, such as components of cell walls,replicases and other enzymes; industrially relevant targets, such asenzymes used in the food industry, reagents intended for research orproduction purposes, and the like.

An “activity”, as referred to herein in connection with the modulationof an activity of a target, can be any activity of the target or anactivity of a molecule which is influenced by the target, which ismodulatable directly or indirectly by a compound or compounds as assayedherein. The activity of the target may be any measurable biological orchemical activity, including binding activity, an enzymatic activity, anactivating or inhibitory activity on a third enzyme or other molecule,the ability to cause disease or influence metabolism or other functions,and the like. Activation and inhibition, as referred to herein, denotethe increase or decrease of a desired activity 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold, 100 fold or more. Where the modulation isinactivation, the inactivation can be substantially completeinactivation. The desired activity may moreover be purely a bindingactivity, which may or may not involve the modulation of the activity ofthe target bound to.

A compound defined herein as “low molecular weight” or a “smallmolecule” is a molecule commonly referred to in the pharmaceutical artsas a “small molecule”. Such compounds are smaller than polypeptides andother, large molecular complexes and can be easily administered to andassimilated by patients and other subjects. Small molecule drugs canadvantageously be formulated for oral administration or intramuscularinjection. For example, a small molecule may have a molecular weight ofup to 2000 Dalton; preferably up to 1000 Dalton; advantageously between250 and 750 Dalton; and more preferably less than 500 Dalton.

A “selectable change” is any change which can be measured and acted uponto identify or isolate the compound which causes it. The selection maytake place at the level of the microcapsule, the microbead, or thecompound itself, optionally when complexed with another reagent. Aparticularly advantageous embodiment is optical detection, in which theselectable change is a change in optical properties, which can bedetected and acted upon for instance in a FACS device to separatemicrocapsules or microbeads displaying the desired change.

As used herein, a change in optical properties refers to any change inabsorption or emission of electromagnetic radiation, including changesin absorbance, luminescence, phosphorescence or fluorescence. All suchproperties are included in the term “optical”. Microcapsules ormicrobeads can be identified and, optionally, sorted, for example, byluminescence, fluorescence or phosphorescence activated sorting. In apreferred embodiment, flow cytometry is employed to identify and,optionally, sort microcapsules or microbeads. A variety of opticalproperties can be used for analysis and to trigger sorting, includinglight scattering (Kerker, 1983) and fluorescence polarisation (Rollandet al., 1985). In a highly preferred embodiment microcapsules ormicrobeads are analysed and, optionally, sorted using a fluorescenceactivated cell sorter (FACS) (Norman, 1930; Mackenzie and Pinder, 1936).

The compounds in microcapsules or on beads can be identified using avariety of techniques familiar to those skilled in the art, includingmass spectroscopy, chemical tagging or optical tagging.

General Techniques

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc. which are incorporated herein by reference)and chemical methods. In addition Harlow & Lane, A Laboratory ManualCold Spring Harbor, N.Y., is referred to for standard ImmunologicalTechniques.

(A) General Description

The microcapsules of the present invention require appropriate physicalproperties to allow the working of the invention.

First, to ensure that the compounds and the target may not diffusebetween microcapsules, the contents of each microcapsule must beisolated from the contents of the surrounding microcapsules, so thatthere is no or little exchange of compounds and target between themicrocapsules over the timescale of the experiment.

Second, the method of the present invention requires that there are onlya limited number of beads per microcapsule. This ensures that thecompounds and the target will be isolated from other beads.

Third, the formation and the composition of the microcapsules must notabolish the activity of the target.

Consequently, any microencapsulation system used must fulfill thesethree requirements. The appropriate system(s) may vary depending on theprecise nature of the requirements in each application of the invention,as will be apparent to the skilled person.

A wide variety of microencapsulation procedures are available (seeBenita, 1996) and may be used to create the microcapsules used inaccordance with the present invention. Indeed, more than 200microencapsulation methods have been identified in the literature(Finch, 1993).

These include membrane enveloped aqueous vesicles such as lipid vesicles(liposomes) (New, 1990) and non-ionic surfactant vesicles (van Hal etal., 1996). These are closed-membranous capsules of single or multiplebilayers of non-covalently assembled molecules, with each bilayerseparated from its neighbour by an aqueous compartment. In the case ofliposomes the membrane is composed of lipid molecules; these are usuallyphospholipids but sterols such as cholesterol may also be incorporatedinto the membranes (New, 1990). A variety of enzyme-catalysedbiochemical reactions, including RNA and DNA polymerisation, can beperformed within liposomes (Chakrabarti et al., 1994; Oberholzer et al.,1995a; Oberholzer et al., 1995b; Walde et al., 1994; Wick & Luisi,1996).

With a membrane-enveloped vesicle system much of the aqueous phase isoutside the vesicles and is therefore non-compartmentalised. Thiscontinuous, aqueous phase should be removed or the biological systems init inhibited or destroyed in order that the reactions are limited to themicrocapsules (Luisi et al., 1987).

Enzyme-catalysed biochemical reactions have also been demonstrated inmicrocapsules generated by a variety of other methods. Many enzymes areactive in reverse micellar solutions (Bru & Walde, 1991; Bru & Walde,1993; Creagh et al., 1993; Haber et al., 1993; Kumar et al., 1989; Luisi& B., 1987; Mao & Walde, 1991; Mao et al., 1992; Perez et al., 1992;Walde et al., 1994; Walde et al., 1993; Walde et al., 1988) such as theAOT-isooctane-water system (Menger & Yamada, 1979).

Microcapsules can also be generated by interfacial polymerisation andinterfacial complexation (Whateley, 1996). Microcapsules of this sortcan have rigid, nonpermeable membranes, or semipermeable membranes.Semipermeable microcapsules bordered by cellulose nitrate membranes,polyamide membranes and lipid-polyamide membranes can all supportbiochemical reactions, including multienzyme systems (Chang, 1987;Chang, 1992; Lim, 1984). Alginate/polylysine microcapsules (Lim & Sun,1980), which can be formed under very mild conditions, have also provento be very biocompatible, providing, for example, an effective method ofencapsulating living cells and tissues (Chang, 1992; Sun et al., 1992).

Non-membranous microencapsulation systems based on phase partitioning ofan aqueous environment in a colloidal system, such as an emulsion, mayalso be used.

Preferably, the microcapsules of the present invention are formed fromemulsions; heterogeneous systems of two immiscible liquid phases withone of the phases dispersed in the other as droplets of microscopic orcolloidal size (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant,1984).

Emulsions may be produced from any suitable combination of immiscibleliquids. Preferably the emulsion of the present invention has water(containing the biochemical components) as the phase present in the formof finely divided droplets (the disperse, internal or discontinuousphase) and a hydrophobic, immiscible liquid (an oil’) as the matrix inwhich these droplets are suspended (the nondisperse, continuous orexternal phase). Such emulsions are termed water-in-oil’ (W/O). This hasthe advantage that the entire aqueous phase containing the biochemicalcomponents is compartmentalised in discreet droplets (the internalphase). The external phase, being a hydrophobic oil, generally containsnone of the biochemical components and hence is inert.

The emulsion may be stabilised by addition of one or more surface-activeagents (surfactants). These surfactants are termed emulsifying agentsand act at the water/oil interface to prevent (or at least delay)separation of the phases. Many oils and many emulsifiers can be used forthe generation of water-in-oil emulsions; a recent compilation listedover 16,000 surfactants, many of which are used as emulsifying agents(Ash and Ash, 1993). Suitable oils include light white mineral oil anddecane. Suitable surfactants include: non-ionic surfactants (Schick,1966) such as sorbitan monooleate (Span™ 80; ICI), sorbitan monostearate(Span™ 60; ICI), polyoxyethylenesorbitan monooleate (Tween™ 80; ICI),and octylphenoxyethoxyethanol (Triton X-100); ionic surfactants such assodium cholate and sodium taurocholate and sodium deoxycholate;chemically inert silicone-based surfactants such aspolysiloxane-polycetyl-polyethylene glycol copolymer (Cetyl DimethiconeCopolyol) (e.g. Abil™ EM90; Goldschmidt); and cholesterol.

Emulsions with a fluorocarbon (or perfluorocarbon) continuous phase(Krafft et al., 2003; Riess, 2002) may be particularly advantageous. Forexample, stable water-in-perfluorooctyl bromide andwater-in-perfluorooctylethane emulsions can be formed using F-alkyldimorpholinophosphates as surfactants (Sadtler et al., 1996).Non-fluorinated compounds are essentially insoluble in fluorocarbons andperfluorocarbons (Curran, 1998; Hildebrand and Cochran, 1949; Hudlicky,1992; Scott, 1948; Studer et al., 1997) and small drug-like molecules(typically <500 Da and Log P<5) (Lipinski et al., 2001) arecompartmentalised very effectively in the aqueous microcapsules ofwater-in-fluorocarbon and water-in-perfluorocarbon emulsions—with littleor no exchange between microcapsules.

Advantageously, compounds can be compartmentalised in microcapsulescomprising non-aqueous (organic) solvents. Non-fluorinated organicsolvents are essentially insoluble and immiscible with fluorocarbons andperfluorocarbons (Curran, 1998; Hildebrand and Cochran, 1949; Hudlicky,1992; Scott, 1948; Studer et al., 1997) allowing the formation ofemulsions with a fluorocarbon (or perfluorocarbon) continuous phase anda discontinous phase formed from a non-aqueous solvent such asdichloromethane, chloroform, carbon tetrachloride, toluene,tetrahydrofuran, diethyl ether, and ethanol. The ability to formsecondary compounds in microcapsules comprising non-aqueous solventsgreatly expands the repertoire of chemical reactions that can beperformed and secondary molecules that can be synthesised therein. Mostof synthetic organic chemistry is carried out in organic solventsincluding dichloromethane, chloroform, carbon tetrachloride, toluene,tetrahydrofuran, diethyl ether, and ethanol. Organic molecules dissolvebetter in organic solvents. Electrostatic interactions are enhanced inorganic solvents (due to the low dielectric constant), whereas they canbe solvated and made less reactive in aqueous solvents. For example,much of contemporary organic chemistry involves reactions relating tocarbonyl chemistry, including the use of metal enolates. Likewise for agrowing number of other organometallic interactions. These reactions areoften carried out under an inert atmosphere in anhydrous solvents(otherwise the reagents would be quenched by water). There are also alarge number of reactions which use palladium catalysis including theSuzuki reaction and the Heck reaction.

Creation of an emulsion generally requires the application of mechanicalenergy to force the phases together. There are a variety of ways ofdoing this which utilise a variety of mechanical devices, includingstirrers (such as magnetic stir-bars, propeller and turbine stirrers,paddle devices and whisks), homogenisers (including rotor-statorhomogenisers, high-pressure valve homogenisers and jet homogenisers),colloid mills, ultrasound and ‘membrane emulsification’ devices (Becher,1957; Dickinson, 1994).

Complicated biochemical processes, notably gene transcription andtranslation are also active in aqueous microcapsules formed inwater-in-oil emulsions. This has enabled compartmentalisation inwater-in-oil emulsions to be used for the selection of genes, which aretranscribed and translated in emulsion microcapsules and selected by thebinding or catalytic activities of the proteins they encode (Doi andYanagawa, 1999; Griffiths and Tawfik, 2003; Lee et al., 2002; Sepp etal., 2002; Tawfik and Griffiths, 1998). This was possible because theaqueous microcapsules formed in the emulsion were generally stable withlittle if any exchange of nucleic acids, proteins, or the products ofenzyme catalysed reactions between microcapsules.

The technology exists to create emulsions with volumes all the way up toindustrial scales of thousands of litres (Becher, 1957; Sherman, 1968;Lissant, 1974; Lissant, 1984).

The preferred microcapsule size will vary depending upon the preciserequirements of any individual screening process that is to be performedaccording to the present invention. In all cases, there will be anoptimal balance between the size of the compound library and thesensitivities of the assays to determine the identity of the compoundand target activity.

The size of emulsion microcapsules may be varied simply by tailoring theemulsion conditions used to form the emulsion according to requirementsof the screening system. The larger the microcapsule size, the larger isthe volume that will be required to encapsulate a given compoundlibrary, since the ultimately limiting factor will be the size of themicrocapsule and thus the number of microcapsules possible per unitvolume.

Water-in-oil emulsions can be re-emulsified to create water-in-oil-inwater double emulsions with an external (continuous) aqueous phase.These double emulsions can be analysed and, optionally, sorted using aflow cytometer (Bernath et al., 2004).

Highly monodisperse microcapsules can be produced using microfluidictechniques. For example, water-in-oil emulsions with less than 3%polydispersity can be generated by droplet break off in a co-flowingsteam of oil (Umbanhowar et al., 2000). Microfluidic systems can also beused for laminar-flow of aqueous microdroplets dispersed in a stream ofoil in microfluidic channels (Thorsen et al., 2001). This allows theconstruction of microfluidic devices for flow analysis and, optionally,flow sorting of microdroplets (Fu et al., 2002).

Microcapsules can, advantageously, be fused or split. For example,aqueous microdroplets can be merged and split using microfluidicssystems (Link et al., 2004; Song et al., 2003). Microcapsule fusionallows the mixing of reagents. Fusion, for example, of a microcapsulecontaining the target with a microcapsule containing the compound couldinitiate the reaction between target and compound. Microcapsulesplitting allows single microcapsules to be split into two or moresmaller microcapsules. For example a single microcapsule containing acompound can be split into multiple microcapsules which can then each befused with a different microcapsule containing a different target. Asingle microcapsule containing a target can also be split into multiplemicrocapsules which can then each be fused with a different microcapsulecontaining a different compound, or compounds at differentconcentrations.

Microcapsules can be optically tagged by, for example, incorporatingfluorochromes. In a preferred configuration, the microcapsules areoptically tagged by incorporating quantum dots: quantum dots of 6colours at 10 concentrations would allow the encoding of 10⁶microcapsules (Han et al., 2001). Microcapsules flowing in an orderedsequence in a microfluidic channel can be encoded (wholly or partially)by their sequence in the stream of microcapsules (positional encoding).

Microbeads, also known by those skilled in the art as microspheres,latex particles, beads, or minibeads, are available in diameters from 20nm to 1 mm and can be made from a variety of materials including silicaand a variety of polymers, copolymers and terpolymers includingpolystyrene (PS), polymethylmethacrylate (PMMA), polyvinyltoluene (PVT),styrene/butadiene (SB) copolymer, and styrene/vinyltoluene (S/VT)copolymer (www.bangslabs.com). They are available with a variety ofsurface chemistries from hydrophobic surfaces (e.g. plain polystyrene),to very hydrophilic surfaces imparted by a wide variety of functionalsurface groups: aldehyde, aliphatic amine, amide, aromatic amine,carboxylic acid, chloromethyl, epoxy, hydrazide, hydroxyl, sulfonate andtosyl. The functional groups permit a wide range of covalent couplingreactions for stable or reversible attachment of compounds to themicrobead surface.

Microbeads can be optically tagged by, for example, incorporatingfluorochromes. For example, one hundred different bead sets have beencreated, each with a unique spectral address due to labelling withprecise ratios of red (>650 nm) and orange (585 nm) fluorochromes(Fulton et al., 1997) (www.luminex.com) and sets of up to 10⁶ beads canbe encoded by incorporating quantum dots of 10 intensities and 6 colours(Han et al., 2001).

The compounds can be connected to the microbeads either covalently ornon-covalently by a variety of means that will be familiar to thoseskilled in the art (see, for example, (Hermanson, 1996)).Advantageously, the compounds are attached via a cleavable linker Avariety of such linkers are familiar to those skilled in the art (seefor example (Gordon and Balasubramanian, 1999)), including for example,linkers which can be cleaved photochemically and reversible covalentbonds which can be controlled by changing the pH (e.g. imines andacylhydrazones), by adjusting the oxido-reductive properties (e.g.disulphides), or using an external catalyst (e.g. cross-metathesis andtransamidation).

The method of the present invention permits the identification ofcompounds which modulate the activity of the target in a desired way inpools (libraries or repertoires) of compounds.

In a highly preferred application, the method of the present inventionis useful for screening libraries of compounds. The inventionaccordingly provides a method according to preceding aspects of theinvention, wherein the compounds are identified from a library ofcompounds.

The compounds identified according to the invention are advantageouslyof pharmacological or industrial interest, including activators orinhibitors of biological systems, such as cellular signal transductionmechanisms suitable for diagnostic and therapeutic applications. In apreferred aspect, therefore, the invention permits the identification ofclinically or industrially useful products. In a further aspect of theinvention, there is provided a product when isolated by the method ofthe invention.

The selection of suitable encapsulation conditions is desirable.Depending on the complexity and size of the compound library to bescreened, it may be beneficial to set up the encapsulation proceduresuch that one or less than one secondary compound is formed permicrocapsule. This will provide the greatest power of resolution. Wherethe library is larger and/or more complex, however, this may beimpracticable; it may be preferable to form several secondary compoundstogether and rely on repeated application of the method of the inventionto identify the desired compound. A combination of encapsulationprocedures may be used to identify the desired compound.

Theoretical studies indicate that the larger the number of compoundscreated the more likely it is that a compound will be created with theproperties desired (see (Perelson and Oster, 1979) for a description ofhow this applies to repertoires of antibodies). It has also beenconfirmed practically that larger phage-antibody repertoires do indeedgive rise to more antibodies with better binding affinities than smallerrepertoires (Griffiths et al., 1994). To ensure that rare variants aregenerated and thus are capable of being identified, a large library sizeis desirable. Thus, the use of optimally small microcapsules isbeneficial.

The largest repertoires of compounds that can be screened in a singleexperiment to date, using two dimensional microarrays of 1 nl volumespots, is ˜10³ (Hergenrother et al., 2000). Using the present invention,at a microcapsule diameter of 2.6 mm (Tawfik and Griffiths, 1998), byforming a three-dimensional dispersion, a repertoire size of at least10¹¹ can be screened using 1 ml aqueous phase in a 20 ml emulsion.

In addition to the compounds, or microbeads coated with compounds,described above, the microcapsules according to the invention willcomprise further components required for the screening process to takeplace. They will comprise the target and a suitable buffer. A suitablebuffer will be one in which all of the desired components of thebiological system are active and will therefore depend upon therequirements of each specific reaction system. Buffers suitable forbiological and/or chemical reactions are known in the art and recipesprovided in various laboratory texts, such as (Sambrook and Russell,2001).

Other components of the system will comprise those necessary forassaying the activity of the target. These may for example comprisesubstrate(s) and cofactor(s) for a reaction catalysed by the target, andligand(s) bound by the target. They may also comprise other catalysts(including enzymes), substrates and cofactors for reactions coupled tothe activity of the target which allow for the activity of the target tobe detected.

(B) Screening Procedures

To screen compounds which bind to or modulate the activity of a target,the target is compartmentalised in microcapsules together with one ormore compounds or compound-coated microbeads. Advantageously eachmicrocapsule contains only a single sort of secondary compound, but manycopies thereof. Advantageously each microbead is coated with only asingle sort of compound, but many copies thereof. Advantageously thecompounds are connected to the microbeads via a cleavable linker,allowing them to be released from the microbeads in the compartments.Advantageously, each microcapsule or microbead is optically tagged toallow identification of the compounds contained within the microcapsuleof attached to the microbead.

(i) Screening Binding

Compounds can be screened directly for binding to a target. In thisembodiment, if the compound is attached to a microbead and has affinityfor the target it will be bound by the target. At the end of thereaction, all of the microcapsules are combined, and all microbeadspooled together in one environment. Microbeads carrying compoundsexhibiting the desired binding can be selected by affinity purificationusing a molecule that specifically binds to, or reacts specificallywith, the target.

In an alternative embodiment, the target can be attached to microbeadsby a variety of means familiar to those skilled in the art (see forexample (Hermanson, 1996)). The compounds to be screened contain acommon feature—a tag. The compounds are released from the microbeads andif the compound has affinity for the target, it will bind to it. At theend of the reaction, all of the microcapsules are combined, and allmicrobeads pooled together in one environment. Microbeads carryingcompounds exhibiting the desired binding can be selected by affinitypurification using a molecule that specifically binds to, or reactsspecifically with, the “tag”.

In an alternative embodiment, microbeads may be screened on the basisthat the compound, which binds to the target, merely hides the ligandfrom, for example, further binding partners. In this eventuality, themicrobead, rather than being retained during an affinity purificationstep, may be selectively eluted whilst other microbeads are bound.

Sorting by affinity is dependent on the presence of two members of abinding pair in such conditions that binding may occur. Any binding pairmay be used for this purpose. As used herein, the term binding pairrefers to any pair of molecules capable of binding to one another.Examples of binding pairs that may be used in the present inventioninclude an antigen and an antibody or fragment thereof capable ofbinding the antigen, the biotin-avidin/streptavidin pair (Savage et al.,1994), a calcium-dependent binding polypeptide and ligand thereof (e.g.calmodulin and a calmodulin-binding peptide (Montigiani et al., 1996;Stofko et al., 1992), pairs of polypeptides which assemble to form aleucine zipper (Tripet et al., 1996), histidines (typicallyhexahistidine peptides) and chelated Cu²⁺, Zn²⁺³⁰ and Ni²⁺, (e.g.Ni-NTA; (Hochuli et al., 1987)), RNA-binding and DNA-binding proteins(Klug, 1995) including those containing zinc-finger motifs (Klug andSchwabe, 1995) and DNA methyltransferases (Anderson, 1993), and theirnucleic acid binding sites.

In an alternative embodiment, compounds can be screened for binding to atarget using a change in the optical properties of the microcapsule orthe microbead.

The change in optical properties of the microcapsule or the microbeadafter binding of the compound to the target may be induced in a varietyof ways, including:

-   -   (1) the compound itself may have distinctive optical properties,        for example, it is fluorescent    -   (2) the optical properties of the compound may be modified on        binding to the target, for example, the fluorescence of the        compound is quenched or enhanced on binding (Voss, 1993; Masui        and Kuramitsu, 1998).    -   (3) the optical properties of the target may be modified on        binding of the compound, for example, the fluorescence of the        target is quenched or enhanced on binding (Guixe et al., 1998;        Qi and Grabowski, 1998)    -   (4) the optical properties of both target and compound are        modified on binding, for example, there can be a fluorescence        resonance energy transfer (FRET) from target to compound (or        vice versa) resulting in emission at the “acceptor” emission        wavelength when excitation is at the “donor” absorption        wavelength (Heim & Tsien, 1996; Mahajan et al., 1998; Miyawaki        et al., 1997).

The invention provides a method wherein a compound with the desiredactivity induces a change in the optical properties of the microcapsule,which enables the microcapsule containing the compound and themicrobeads contained therein to be identified, and optionally, sorted.

In an alternative embodiment, the invention provides a method whereinmicrobeads are analysed following pooling of the microcapsules into oneor more common compartments. In this embodiment, a compound having thedesired activity modifies the optical properties of the microbead whichcarried it (and which resides in the same microcapsule) to allow it tobe identified, and optionally, sorted.

In this embodiment, it is not necessary for binding of the compound tothe target to directly induce a change in optical properties.

In this embodiment, if the compound attached to the microbead hasaffinity for the target it will be bound by the target. At the end ofthe reaction, all of the microcapsules are combined, and all microbeadspooled together in one environment. Microbeads carrying compoundsexhibiting the desired binding can be identified by adding reagents thatspecifically bind to, or react specifically with, the target and therebyinduce a change in the optical properties of the microbeads allowingtheir identification. For example, a fluorescently-labelled anti-targetantibody can be used, or an anti-target antibody followed by a secondfluorescently labelled antibody which binds the first.

In an alternative embodiment, the target can be attached to themicrobeads by a variety of means familiar to those skilled in the art(see for example (Hermanson, 1996)). The compounds to be screenedcontain a common feature—a tag. The compounds are released from themicrobeads and if the compound has affinity for the target, it will bindto it. At the end of the reaction, all of the microcapsules arecombined, and all microbeads pooled together in one environment.Microbeads carrying compounds exhibiting the desired binding can beidentified by adding reagents that specifically bind to, or reactspecifically with, the “tag” and thereby induce a change in the opticalproperties of the microbeads allowing their identification. For example,a fluorescently-labelled anti-“tag” antibody can be used, or ananti-“tag” antibody followed by a second fluorescently labelled antibodywhich binds the first.

In an alternative embodiment, microbeads may be identified on the basisthat the gene product, which binds to the ligand, merely hides theligand from, for example, further binding partners which would otherwisemodify the optical properties of the microbeads. In this case microbeadswith unmodified optical properties would be selected.

Fluorescence may be enhanced by the use of Tyramide Signal Amplification(TSA™) amplification to make the microbeads fluorescent (Sepp et al.,2002). This involves peroxidase (linked to another compound) binding tothe microbeads and catalysing the conversion of fluorescein-tyramine into a free radical form which then reacts (locally) with the microbeads.Methods for performing TSA are known in the art, and kits are availablecommercially from NEN.

TSA may be configured such that it results in a direct increase in thefluorescence of the microbeads, or such that a ligand is attached to themicrobeads which is bound by a second fluorescent molecule, or asequence of molecules, one or more of which is fluorescent.

(ii) Screening for Regulation of Binding

In an alternative embodiment, the invention can be used to screencompounds which act to regulate a biochemical process. If the compoundactivates a binding activity of a target, a ligand for the target whichis activated can be attached to microbeads by a variety of meansfamiliar to those skilled in the art (see for example (Hermanson,1996)). At the end of the reaction, all of the microcapsules arecombined, and all microbeads pooled together in one environment.Microbeads carrying compounds exhibiting the desired binding can beselected by affinity purification using a molecule that specificallybinds to, or reacts specifically with, the target.

In an alternative embodiment, microbeads may be screened on the basisthat the compound inhibits the binding activity of a target. In thiseventuality, the microbead, rather than being retained during anaffinity purification step, may be selectively eluted whilst othermicrobeads are bound.

In an alternative embodiment, compounds can be screened for the abilityto modulates a binding activity of a target using a change in theoptical properties of the microcapsule or the microbead.

The change in optical properties of the microcapsule or the microbeadafter binding of the target to its ligand may be induced in a variety ofways, including:

-   -   (1) the ligand itself may have distinctive optical properties,        for example, it is fluorescent    -   (2) the optical properties of the ligand may be modified on        binding to the target, for example, the fluorescence of the        ligand is quenched or enhanced on binding (Voss, 1993; Masui and        Kuramitsu, 1998).    -   (3) the optical properties of the target may be modified on        binding of the ligand, for example, the fluorescence of the        target is quenched or enhanced on binding (Guixe et al., 1998;        Qi and Grabowski, 1998)    -   (4) the optical properties of both target and ligand are        modified on binding, for example, there can be a fluorescence        resonance energy transfer (FRET) from target to ligand (or vice        versa) resulting in emission at the “acceptor” emission        wavelength when excitation is at the “donor” absorption        wavelength (Heim & Tsien, 1996; Mahajan et al., 1998; Miyawaki        et al., 1997).

The invention provides a method wherein a compound with the desiredactivity induces a change in the optical properties of the microcapsule,which enables the microcapsule containing the compound and themicrobeads contained therein to be identified, and optionally, sorted.

In an alternative embodiment, the invention provides a method whereinmicrobeads are analysed following pooling of the microcapsules into oneor more common compartments. In this embodiment, a compound having thedesired activity modifies the optical properties of the microbead whichcarried it (and which resides in the same microcapsule) to allow it tobe identified, and optionally, sorted.

In this embodiment, it is not necessary for binding of the target to theligand to directly induce a change in optical properties.

In this embodiment, if a ligand attached to the microbead has affinityfor the target it will be bound by the target. At the end of thereaction, all of the microcapsules are combined, and all microbeadspooled together in one environment. Microbeads carrying compounds whichmodulate the binding activity can be identified by adding reagents thatspecifically bind to, or react specifically with, the target and therebyinduce a change in the optical properties of the microbeads allowingtheir identification. For example, a fluorescently-labelled anti-targetantibody can be used, or an anti-target antibody followed by a secondfluorescently labelled antibody which binds the first.

In an alternative embodiment, the target can be attached to themicrobeads by a variety of means familiar to those skilled in the art(see for example (Hermanson, 1996)). The ligand to be screened containsa feature—a tag. At the end of the reaction, all of the microcapsulesare combined, and all microbeads pooled together in one environment.Microbeads carrying compounds which modulate binding can be identifiedby adding reagents that specifically bind to, or react specificallywith, the “tag” and thereby induce a change in the optical properties ofthe microbeads allowing their identification. For example, afluorescently-labelled anti-“tag” antibody can be used, or an anti-“tag”antibody followed by a second fluorescently labelled antibody whichbinds the first.

Fluorescence may be enhanced by the use of Tyramide Signal Amplification(TSA™) amplification to make the microbeads fluorescent (Sepp et al.,2002), as above.

(iii) Screening for Regulation of Catalysis

In an alternative embodiment, the invention provides a method wherein acompound with the desired activity induces a change in the opticalproperties of the microcapsule, which enables the microcapsulecontaining the compound and, optionally, the microbeads containedtherein to be identified, and optionally, sorted. The optical propertiesof microcapsules can be modified by either:

-   -   (a) the substrate and product of the regulated reaction having        different optical properties (many fluorogenic enzyme substrates        are available commercially, see for example (Haugland, 1996 and        www.probes.com) including substrates for glycosidases,        phosphatases, peptidases and proteases, or    -   (b) the presence of reagents which specifically bind to, or        react with, the product (or substrate) of the regulated reaction        in the microcapsule and which thereby induce a change in the        optical properties of the microcapsules allowing their        identification.

A wide range of assays for screening libraries of compounds for thosewhich modulate the activity of a target are based on detecting changesin optical properties and can be used to screen compounds according tothis invention. Such assays are well known to those skilled in the art(see for example Haugland, 1996 and www.probes.com).

Alternatively, selection may be performed indirectly by coupling a firstreaction to subsequent reactions that takes place in the samemicrocapsule. There are two general ways in which this may be performed.First, the product of the first reaction could be reacted with, or boundby, a molecule which does not react with the substrate(s) of the firstreaction. A second, coupled reaction will only proceed in the presenceof the product of the first reaction. A regulatory compound can then beidentified by the properties of the product or substrate of the secondreaction.

Alternatively, the product of the reaction being selected may be thesubstrate or cofactor for a second enzyme-catalysed reaction. The enzymeto catalyse the second reaction can be incorporated in the reactionmixture prior to microencapsulation. Only when the first reactionproceeds will the coupled enzyme generate an identifiable product.

This concept of coupling can be elaborated to incorporate multipleenzymes, each using as a substrate the product of the previous reaction.This allows for selection of regulators of enzymes that will not reactwith an immobilised substrate. It can also be designed to give increasedsensitivity by signal amplification if a product of one reaction is acatalyst or a cofactor for a second reaction or series of reactionsleading to a selectable product (for example, see (Johannsson, 1991;Johannsson and Bates, 1988). Furthermore an enzyme cascade system can bebased on the production of an activator for an enzyme or the destructionof an enzyme inhibitor (see (Mize et al., 1989)). Coupling also has theadvantage that a common screening system can be used for a whole groupof enzymes which generate the same product and allows for the selectionof regulation of complicated multi-step chemical transformations andpathways.

In an alternative embodiment, if the target is itself an enzyme, orregulates a biochemical process which is enzymatic, the microbead ineach microcapsule may be coated with the substrate for the enzymaticreaction. The regulatory compound will determine the extent to which thesubstrate is converted into the product. At the end of the reaction themicrobead is physically linked to the product of the catalysed reaction.When the microcapsules are combined and the reactants pooled, microbeadswhich were coated with activator compounds can be identified by anyproperty specific to the product. If an inhibitor is desired, selectioncan be for a chemical property specific to the substrate of theregulated reaction.

It may also be desirable, in some cases, for the substrate not to beattached to the microbead. In this case the substrate would contain aninactive “tag” that requires a further step to activate it such asphotoactivation (e.g. of a “caged” biotin analogue, (Pirrung and Huang,1996; Sundberg et al., 1995)). After convertion of the substrate toproduct the “tag” is activated and the “tagged” substrate and/or productbound by a tag-binding molecule (e.g. avidin or streptavidin) attachedto the microbead. The ratio of substrate to product attached to thenucleic acid via the “tag” will therefore reflect the ratio of thesubstrate and product in solution. A substrate tagged with caged biotinhas been used to select for genes encoding enzymes withphosphotriesterase activity using a procedure based oncompartmentalisation in microcapsules (Griffiths and Tawfik, 2003). Thephosphotriesterase substrate was hydrolysed in solution in microcapsulescontaining active enzyme molecules, and after the reaction wascompleted, the caging group was released by irradiation to allow theproduct to bind, via the biotin moiety, to microbeads to which the geneencoding the enzyme was attached.

After the microbeads and the contents of the microcapsules are combined,those microbeads coated with regulators can be selected by affinitypurification using a molecule (e.g. an antibody) that binds specificallyto the product or substrate as appropriate.

In an alternative embodiment, the invention provides a method whereinthe microbeads are analysed following pooling of the microcapsules intoone or more common compartments. Microbeads coated with regulatorcompounds can be identified using changes in optical properties of themicrobeads. The optical properties of microbeads with product (orsubstrate) attached can be modified by either:

-   -   (1) the product-microbead complex having characteristic optical        properties not found in the substrate-microbead complex, due to,        for example;        -   (a) the substrate and product having different optical            properties (many fluorogenic enzyme substrates are available            commercially (see for example Haugland, 1996 and            www.probes.com) including substrates for glycosidases,            phosphatases, peptidases and proteases, or        -   (b) the substrate and product having similar optical            properties, but only the product, and not the substrate            binds to, or reacts with, the microbead;    -   (2) adding reagents which specifically bind to, or react with,        the product (or substrate) and which thereby induce a change in        the optical properties of the microbeads allowing their        identification (these reagents can be added before or after        breaking the microcapsules and pooling the microbeads). The        reagents;        -   (a) bind specifically to, or react specifically with, the            product, and not the substrate, (or vice versa) if both            substrate and product are attached to the microbeads, or        -   (b) optionally bind both substrate and product if only the            product, and not the substrate binds to, or reacts with, the            microbeads (or vice versa).

In this scenario, the substrate (or one of the substrates) can bepresent in each microcapsule unlinked to the microbead, but has amolecular “tag” (for example biotin, DIG or DNP or a fluorescent group).When the regulated enzyme converts the substrate to product, the productretains the “tag” and is then captured in the microcapsule by theproduct-specific antibody. When all reactions are stopped and themicrocapsules are combined, these microbeads will be “tagged” and mayalready have changed optical properties, for example, if the “tag” was afluorescent group. Alternatively, a change in optical properties of“tagged” microbeads can be induced by adding a fluorescently labelledligand which binds the “tag” (for example fluorescently-labelledavidin/streptavidin, an anti-“tag” antibody which is fluorescent, or anon-fluorescent anti-“tag” antibody which can be detected by a secondfluorescently-labelled antibody).

(iv) Screening for Compound Specificity/Selectivity

Compounds with specificity or selectivity for certain targets and notothers can be specifically identified by carrying out a positive screenfor regulation of a reaction using one substrate and a negative screenfor regulation of a reaction with another substrate. For example, twosubstrates, specific for two different target enzymes, are each labelledwith different fluorogenic moieties. Each target enzymes catalyse thegeneration of a product with a different fluorescence spectrum resultingin different optical properties of the microcapsules depending on thespecificity of the compound for two targets.

(v) Screening Using Cells

In the current drug discovery paradigm, validated recombinant targetsform the basis of in vitro high-throughput screening (HTS) assays.Isolated proteins cannot, however, be regarded as representative ofcomplex biological systems; hence, cell-based systems can providegreater confidence in compound activity in an intact biological system.A wide range of cell-based assays for drug leads are known to thoseskilled in the art. Cells can be compartmentalised in microcapsules,such as the aqueous microdroplets of a water-in-oil emulsion (Ghadessy,2001). The effect of a compound(s) on a target can be determined bycompartmentalising a cell (or cells) in a microcapsule together with acompound(s) and using an appropriate cell-based assay to identify thosecompartments containing compounds with the desired effect on thecell(s). The use of water-in-fluorocarbon emulsions may be particularlyadvantageous: the high gas dissolving capacity of fluorocarbons cansupport the exchange of respiratory gases and has been reported to bebeneficial to cell culture systems (Lowe, 2002).

(vi) Flow Cytometry

In a preferred embodiment of the invention the microcapsules ormicrobeads will be analysed and, optionally, sorted by flow cytometry.Many formats of microcapsule can be analysed and, optionally, sorteddirectly using flow cytometry. Some formats of microcapsule may requirethat the microcapsules be further processed before analysis or sorting.For example, water-in-oil emulsions can be converted intowater-in-oil-in-water double emulsions to facilitate analysis by flowcytometry (Bernath et al., 2004). Multiple emulsions are prepared by there-emulsification of a simple primary water-in-oil (or oil-in-water)emulsion to provide water-in-oil-in-water (or oil-in-water-in-oil)emulsions (Davis and Walker, 1987).

Highly monodisperse microcapsules can be produced using microfluidictechniques. For example, water-in-oil emulsions with less than 3%polydispersity can be generated by droplet break off in a co-flowingsteam of oil (Umbanhowar, 2000). Microfluidic systems can also be usedfor laminar-flow of aqueous microdroplets dispersed in a stream of oilin microfluidic channels (Thorsen, 2001). This allows the constructionof microfluidic devices for flow analysis and, optionally, flow sortingof microdroplets (Fu, 2002).

A variety of optical properties can be used for analysis and to triggersorting, including light scattering (Kerker, 1983) and fluorescencepolarisation (Rolland et al., 1985). In a highly preferred embodimentthe difference in optical properties of the microcapsules or microbeadswill be a difference in fluorescence and, if required, the microcapsulesor microbeads will be sorted using a fluorescence activated cell sorter(Norman, 1980; Mackenzie and Pinder, 1986), or similar device. Flowcytometry has a series of advantages:

-   -   (1) commercially available fluorescence activated cell sorting        equipment from established manufacturers (e.g. Becton-Dickinson,        Coulter, Cytomation) allows the analysis and sorting at up to        100,000 microcapsules or microbeads s⁻¹.    -   (2) the fluorescence signal from each microcapsule or microbead        corresponds tightly to the number of fluorescent molecules        present. As little as few hundred fluorescent molecules per        microcapsules or microbeads can be quantitatively detected;    -   (3) the wide dynamic range of the fluorescence detectors        (typically 4 log units) allows easy setting of the stringency of        the sorting procedure, thus allowing the recovery of the optimal        number microcapsules or microbeads from the starting pool (the        gates can be set to separate microcapsules or microbeads with        small differences in fluorescence or to only separate out        microcapsules or microbeads with large differences in        fluorescence, dependant on the selection being performed);    -   (4) commercially available fluorescence-activated cell sorting        equipment can perform simultaneous excitation and detection at        multiple wavelengths (Shapiro, 1995) allowing positive and        negative selections to be performed simultaneously by monitoring        the labelling of the microcapsules or microbeads with two to        thirteen (or more) fluorescent markers, for example, if        substrates for two alternative targets are labelled with        different fluorescent tags the microcapsules or microbeads can        labelled with different fluorophores dependent on the target        regulated.

If the microcapsules or microbeads are optically tagged, flow cytometrycan also be used to identify the compound or compounds in themicrocapsule or coated on the microbeads (see below). Optical taggingcan also be used to identify the concentration of the compound in themicrocapsule (if more than one concentration is used in a singleexperiment) or the number of compound molecules coated on a microbead(if more than one coating density is used in a single experiment).Furthermore, optical tagging can be used to identify the target in amicrocapsule (if more than one target is used in a single experiment).This analysis can be performed simultaneously with measuring activity,after sorting of microcapsules containing microbeads, or after sortingof the microbeads.

(vii) Microcapsule Identification and Sorting

The invention provides for the identification and, optionally, thesorting of intact microcapsules where this is enabled by the sortingtechniques being employed. Microcapsules may be identified and,optionally, sorted as such when the change induced by the desiredcompound either occurs or manifests itself at the surface of themicrocapsule or is detectable from outside the microcapsule. The changemay be caused by the direct action of the compound, or indirect, inwhich a series of reactions, one or more of which involve the compoundhaving the desired activity leads to the change. For example, where themicrocapsule is a membranous microcapsule, the microcapsule may be soconfigured that a component or components of the biochemical systemcomprising the target are displayed at its surface and thus accessibleto reagents which can detect changes in the biochemical system regulatedby the compound on the microbead within the microcapsule.

In a preferred aspect of the invention, however, microcapsuleidentification and, optionally, sorting relies on a change in theoptical properties of the microcapsule, for example absorption oremission characteristics thereof, for example alteration in the opticalproperties of the microcapsule resulting from a reaction leading tochanges in absorbance, luminescence, phosphorescence or fluorescenceassociated with the microcapsule. All such properties are included inthe term “optical”. In such a case, microcapsules can be identified and,optionally, sorted by luminescence, fluorescence or phosphorescenceactivated sorting. In a highly preferred embodiment, flow cytometry isemployed to analyse and, optionally, sort microcapsules containingcompounds having a desired activity which result in the production of afluorescent molecule in the microcapsule.

In an alternative embodiment, a change in microcapsule fluorescence,when identified, is used to trigger the modification of the microbeadwithin the compartment. In a preferred aspect of the invention,microcapsule identification relies on a change in the optical propertiesof the microcapsule resulting from a reaction leading to luminescence,phosphorescence or fluorescence within the microcapsule. Modification ofthe microbead within the microcapsules would be triggered byidentification of luminescence, phosphorescence or fluorescence. Forexample, identification of luminescence, phosphorescence or fluorescencecan trigger bombardment of the compartment with photons (or otherparticles or waves) which leads to modification of the microbead ormolecules attached to it. A similar procedure has been describedpreviously for the rapid sorting of cells (Keij et al., 1994).Modification of the microbead may result, for example, from coupling amolecular “tag”, caged by a photolabile protecting group to themicrobeads: bombardment with photons of an appropriate wavelength leadsto the removal of the cage. Afterwards, all microcapsules are combinedand the microbeads pooled together in one environment. Microbeads coatedwith compounds exhibiting the desired activity can be selected byaffinity purification using a molecule that specifically binds to, orreacts specifically with, the “tag”.

(C) Compound Libraries

(i) Primary Compounds Libraries

Libraries of primary compounds can be obtained from a variety ofcommercial sources. The compounds in the library can be made by avariety of means well known to those skilled in the art. Optionally,compound libraries can be made by combinatorial synthesis usingspatially resolved parallel synthesis or using split synthesis,optionally to generate one-bead-one-compound libraries. The compoundscan, optionally, be synthesised on beads. These beads can becompartmentalised in microcapsules directly or the compounds releasedbefore compartmentalisation.

Advantageously, only a single type of compound, but multiple copiesthereof is present in each microcapsule.

The compounds can, optionally, be connected to microbeads eithercovalently or non-covalently by a variety of means that will be familiarto those skilled in the art (see, for example, (Hermanson, 1996)).

Microbeads are available with a variety of surface chemistries fromhydrophobic surfaces (e.g. plain polystyrene), to very hydrophilicsurfaces imparted by a wide variety of functional surface groups:aldehyde, aliphatic amine, amide, aromatic amine, carboxylic acid,chloromethyl, epoxy, hydrazide, hydroxyl, sulfonate and tosyl. Thefunctional groups permit a wide range of covalent coupling reactions,well known to those skilled in the art, for stable or reversibleattachment of compounds to the microbead surface.

Advantageously, the compounds are attached to the microbeads via acleavable linker. A variety of such linkers are familiar to thoseskilled in the art (see for example (Gordon and Balasubramanian, 1999)),including for example, linkers which can be cleaved photochemically andreversible covalent bonds which can be controlled by changing the pH(e.g. imines and acylhydrazones), by adjusting the oxido-reductiveproperties (e.g. disulphides), or using an external catalyst (e.g.cross-metathesis and transamidation).

Advantageously, only a single type of compound, but multiple copiesthereof is attached to each bead.

(ii) Second Compound Libraries

Secondary compound libraries are created by reactions between primarycompounds in microcapsules. Secondary compounds can be created by avariety of two component, and multi-component reactions well known tothose skilled in the art (Armstrong et al., 1996; Domling, 2002; Domlingand Ugi, 2000; Ramstrom and Lehn, 2002).

To form secondary compound libraries by a two-component reaction, twosets of compounds are compartmentalised in microcapsules such that manycompartments contain two or more compounds. Advantageously, the modalnumber of compounds per microcapsule is two. Advantageously, themicrocapsules contain at least one type of compound from each set ofcompounds. Advantageously, the microcapsules contain one type ofcompound from each set of compounds. The secondary compounds are formedby chemical reactions between primary compounds from different sets. Thesecondary compound may be the result of a covalent or non-covalentreaction between the primary compounds.

A variety of chemistries, familiar to those skilled in the art, aresuitable to form secondary compounds in two-component reactions. Forexample, reversible covalent bonds which can be controlled by changingthe pH (e.g. imines and acylhydrazones), by adjusting theoxido-reductive properties (e.g. disulphides), or using an externalcatalyst (e.g. cross-metathesis and transamidation), can be used(Ramstrom and Lehn, 2002).

In a further embodiment, the method can also be used to create secondarycompound libraries using three-component, four-component and higherorder multi-component reactions. Three, four or more sets of compounds(as appropriate) are compartmentalised in microcapsules. The compoundsare compartmentalised in microcapsules such that many compartmentscontain multiple compounds. Advantageously, the modal number ofcompounds per microcapsule is equal to the number of components in thereaction. Advantageously, the microcapsules contain at least one type ofcompound from each set of compounds. Advantageously, the microcapsulescontain one type of compound from each set of compounds. The secondarycompounds are formed by chemical reactions between primary compoundsfrom different sets. The secondary compound may be the result ofcovalent or non-covalent reactions between the primary compounds.

Examples of suitable multi-component reactions are the Strecker,Hantzsch, Biginelli, Mannich, Passerini, Bucherer-Bergs and Pauson-Khandthree-component reactions and the Ugi four-component reaction (Armstronget al., 1996; Domling, 2002; Domling and Ugi, 2000).

Secondary compound libraries may also be built using a scaffold moleculewhich is common to all the secondary compounds (Ramstrom and Lehn,2002). This scaffold molecule may be compartmentalised intomicrocapsules together with the other primary compounds.

In a further embodiment, to form secondary compound libraries by atwo-component reaction, two sets of compounds are attached tomicrobeads, advantageously to give only a single type of molecule permicrobead. The microbeads are compartmentalised in microcapsules suchthat many compartments contain two or more microbeads. Advantageously,the modal number of beads per microcapsule is two. The compoundscomprising at least one of the two sets are released from themicrobeads. The secondary compounds are formed by chemical reactionsbetween primary compounds from different sets. The secondary compoundmay be the result of a covalent or non-covalent reaction between theprimary compounds.

In a further embodiment, the method can also be used to create secondarycompound libraries using three-component, four-component and higherorder multi-component reactions. Three, four or more sets of compounds(as appropriate) are attached to microbeads, advantageously to give onlya single type of molecule per microbead. The microbeads arecompartmentalised in microcapsules such that many compartments containmultiple microbeads. Advantageously, the modal number of beads permicrocapsule is equal to the number of components in the reaction. Thecompounds comprising either all, or all bar one, of the sets arereleased from the microbeads. The secondary compounds are formed bychemical reactions between primary compounds from different sets. Thesecondary compound may be the result of covalent or non-covalentreactions between the primary compounds.

Advantageously, the same reversible covalent bond can used to couple theprimary compound to the microbead as is used to form the secondarycompound.

Secondary compound libraries may also be built using a scaffold moleculewhich is common to all the secondary compounds (Ramstrom and Lehn,2002). This scaffold molecule may be compartmentalised intomicrocapsules together with the microbeads.

(D) Identification Compounds

The compounds in microcapsules or on microbeads can be identified in avariety of ways. If the identified microcapsules are sorted (e.g. byusing a fluorescence activated cell sorter—FACS) the compounds can beidentified by direct analysis, for example by mass-spectroscopy. If thecompounds remain attached to beads isolated as a result of selection(for example by affinity purification) or sorting (for example using aFACS) they can also be identified by direct analysis, for example bymass-spectroscopy. The microcapsules or beads can also be tagged by avariety of means well known to those skilled in the art and the tag usedto identify the compound attached to the beads (Czarnik, 1997).Chemical, spectrometric, electronic, and physical methods to encode thecompounds may all be used. In a preferred embodiment microcapsules orbeads have different optical properties and are thereby opticallyencoded. In a preferred embodiment encoding is based on microcapsules orbeads having different fluorescence properties. In a highly preferredembodiment the microcapsules or beads are encoded using fluorescentquantum dots present at different concentrations in the microcapsule orbead (Han, 2001). Microcapsules flowing in an ordered sequence in amicrofluidic channel can also be encoded (wholly or partially) by theirsequence in the stream of microcapsules (positional encoding).

Advantageously, each compounds is present in different microcapsules atdifferent concentrations (typically at concentrations varying from mM tonM) allowing the generation of a dose-response curve. This would, forexample, allow the determination of the inhibition constant (K_(i)) ofan inhibitory compound. The concentration of the compounds in themicrocapsules can be determined by, for example, optical encoding orpositional encoding of the microcapsules or microbeads as above.

(E) Identification of Targets

Advantageously, multiple different targets can be compartmentalised inmicrocapsules such that each microcapsule contains multiple copies ofthe same target. For example, multiple protein kinases, or multiplepolymorphic variants of a single target, can be compartmentalised toallow the specificity of compounds to be determined. The identity of thetarget in a microcapsule can be determined by, for example, opticalencoding or positional encoding of the microcapsules or microbeads asabove.

Expressed in an alternative manner, there is provided a method for thesynthesis and identification of compounds which bind to a targetcomponent of a biochemical system or modulate the activity of thetarget, comprising the steps of:

-   -   (a) compartmentalising two or more sets of primary compounds        into microcapsules together with the target such that many        compartments contain two or more primary compounds;    -   (b) forming secondary compounds in the microcapsules by chemical        reactions between primary compounds from different sets; and    -   (c) identifying subsets of primary compounds which react to form        secondary compounds which bind to or modulate the activity of        the target.

There is also provided a method for the synthesis and identification ofcompounds which bind to a target component of a biochemical system ormodulate the activity of the target, comprising the steps of:

-   -   (1) attaching two or more sets of primary compounds onto        microbeads;    -   (2) compartmentalising the microbeads into microcapsules        together with the target such that many compartments contain two        or more microbeads;    -   (3) releasing the primary compounds from the microbeads;    -   (4) forming secondary compounds in the microcapsules by chemical        reactions between primary compounds from different sets; and    -   (5) identifying subsets of primary compounds which react to form        secondary compounds which bind to or modulate the activity of        the target.

If the primary compounds react, not only with other primary compounds inthe same compartment, but also with other microbeads in the compartment,the primary compounds which react together to form a secondary compoundcan be identified by direct analysis of the compounds present on amicrobeads isolated as a result of selection or sorting. For example, ifthe primary compounds are linked to the beads via a disulphide bond whenthey are released in the compartment the primary compounds will reactboth with each other to form a secondary compound and with thesulphydryl groups on the beads. Hence, if two beads areco-compartmentalised, each bead will end up carrying both primarycompounds. After isolation of these beads both primary compounds whichreacted to form the secondary compound can be identified.

Various aspects and embodiments of the present invention are illustratedin the following examples. It will be appreciated that modification ofdetail may be made without departing from the scope of the invention.

Examples Example 1

Screening for Inhibitors of the Enzyme Protein Tyrosine Phosphatase 1B(PTP1B)

PTP1B is a negative regulator of insulin and leptin signal transduction.Resistance to insulin and leptin are hallmarks of type 2 diabetesmellitus and obesity and hence PTP1B is an attractive drug target fordiabetes and obesity therapy (Johnson et al., 2002). Two water-in-oilemulsions are made as follows.

A solution of 1% (w/v) Span 60 and 1% (w/v) cholesterol in decane (allfrom Sigma Aldrich) is prepared by dissolving 80 mg of Span 60 and 80 mgof cholesterol into 7.84 ml of decane. The decane is heated to 45° C. toallow complete solubilization of the surfactant and cholesterol. Thesurfactant/decane solution is divided over batches of 200 μl and placedin a block-heater at 37° C.

A hand-extruding device (Mini extruder, Avanti Polar Lipids Inc,Alabaster, Ala., USA) is assembled according to the manufacturer'sinstructions. For extrusion, a single 19 mm Track-Etch polycarbonatefilter with average pore size of 14 μm (Whatman Nuclepore, Whatman,Maidstone, UK) is fitted inside the mini extruder. Two gas-tight 1 mlHamilton syringes (Gastight #1001, Hamilton Co, Reno, Nev., USA) areused for extrusion. The extruder was pre-rinsed with 3×1 ml of decane byloading one of the Hamilton syringes with 1 ml of decane, placing thesyringe at one and of the mini extruder and extruding it through thefilters into the empty Hamilton syringe on the other side of theextruder.

The first emulsion is made by loading 50 μl of 100 μM compound 2 (FIG.1), which has a bis-difluoromethylene phosphonate and is a known PTP1Binhibitor (Johnson et al., 2002), the target enzyme (human recombinantPTP 1 B, residues 1-322; Biomol Research Laboratories, Inc.) at 5 mU/ml,the fluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP) (Molecular Probes), and 100 μM Texas Red (Sigma;excitation/emmission maxima 595/615 nm; red fluorescence) in a buffercompatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10%glycerol, 1 mM EDTA) (Doman et al., 2002) into one of the Hamiltonsyringes, and 200 μl of the pre-heated decane/surfactant mix into theother Hamilton syringe. The syringes are fitted into the openings onboth sides of the filter holder of the extruder. The compound mix isforced through the filter holder into the alternate syringe containingthe decane/surfactant mix and directly forced back into the originalsyringe to complete one round of extrusion. In total, 7.5 rounds ofextrusion are completed. The filled syringe is removed from the extruderand emptied into a 1.7 ml Axygen tube (#MCT-175-C, Axygen Scientific,Inc., Union City, Calif., USA).

A second water-in-oil emulsion is made identical to the emulsion abovebut containing 100 μM hydrocinnamic acid (Aldrich), a compound that isnot a PTP1B inhibitor, in place of compound 2, and 100 μM calcein(Sigma; excitation/emmission maxima 470/509 nm; green fluorescence) inplace of Texas Red.

The two emulsions are mixed by vortexing in ratios varying from 1:1000to 1:1 (compound 2 emulsion: hydrocinnamic acid emulsion) and incubatedat 37° C. for 30 min. Inhibitors reduce the amount of non-fluorescentsubstrate (DiFMUP) converted to the dephosphorylated product (DiFMU;excitation/emmission maxima 358/452 nm; blue fluorescence).

The water-in-oil emulsions are then converted into water-in-oil-in waterdouble emulsions as follows. The extruder (see above) is disassembled,cleaned extensively with soap and reversed-osmosis water, andre-assembled. A single 19 mm Track-Etch polycarbonate filter with anaverage pore size of 8 μm is fitted. The extruder is pre-rinsed with 3×1ml phosphate-buffered saline solution (PBS). 750 μl of PBS containing0.5% (w/v) Tween 80 (Sigma Aldrich) is loaded into a 1 ml gas-tightHamilton syringe and fitted into the extruder. 250 μl of thewater-in-oil emulsion is loaded into the alternate 1 ml Hamilton syringeand fitted into the extruder. The emulsion is forced through the filterinto the alternate syringe containing the PBS/0.5% Tween 80 andimmediately forced back into the original syringe to complete one cycleof extrusion. In total, 4.5 cycles of extrusion are performed. Thefilled syringe is removed from the extruder and emptied into a 1.7 mlAxygen tube. The water-in-oil-in-water double emulsions formed areplaced on ice.

The double emulsions are then analysed by multi-colour flow cytometeryusing a MoFlo (Cytomation) flow cytometer. Predominantly, microcapsulesexhibiting green fluorescence (and containing hydrocinnamic acid) alsoshow blue fluorescence due to dephosphorylation of DiFMUP by PTP1B.Predominantly, microcapsules exhibiting red fluorescence (and containingcompound 2) also show little or no blue fluorescence due to inhibitionof PTP1B.

Example 2

Two aqueous mixtures are made on ice (to prevent reaction). The firstmixture contains 100 μM compound 2 (FIG. 1), which has abis-difluoromethylene phosphonate and is a known PTP1B inhibitor(Johnson et al., 2002), the target enzyme (human recombinant PTP1B,residues 1-322; Biomol Research Laboratories, Inc.) at 5 mU/ml, thefluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferyl phosphate(DiFMUP) (Molecular Probes), and 100 μM Texas Red (Sigma;excitation/emmission maxima 595/615 nm; red fluorescence) in a buffercompatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10%glycerol, 1 mM EDTA) (Doman et al., 2002). A second mixture is createdidentical to the above but containing 100 μM hydrocinnamic acid(Aldrich), a compound that is not a PTP1B inhibitor, in place ofcompound 2, and 100 μM calcein (Sigma; excitation/emmission maxima470/509 nm; green fluorescence) in place of Texas Red.

50 μl of each of the compound mixtures is added sequentially to asolution of 1% (w/v) Span 60 and 1% (w/v) cholesterol in decane, madeand held at 37° C. as example 1, whilst homogenising at 25,000 r.p.m.using an Ultra-Turrax T8 Homogenizer (IKA) with a 5 mm dispersing tool.Homogenisation is continued for 3 minutes after the addition of thesecond aliquot. The coarse emulsion produced is then extruded as inexample 1 to create a fine water-in-oil emulsion and incubated at 37° C.for 30 min. Inhibitors reduce the amount of non-fluorescent substrate(DiFMUP) converted to the dephosphorylated product (DiFMU;excitation/emmission maxima 358/452 nm; blue fluorescence). Thewater-in-oil emulsion is then converted into a water-in-oil-in waterdouble emulsion and analysed by multi-colour flow cytometery as inexample 1. Predominantly, microcapsules exhibiting green fluorescence(and containing hydrocinnamic acid) also show blue fluorescence due todephosphorylation of DiFMUP by PTP1B. Predominantly, microcapsulesexhibiting red fluorescence (and containing compound 2) also show littleor no blue fluorescence due to inhibition of PTP1B.

Example 3

Screening of PTP1B Inhibitors from a Compound Library

100 water-in-oil emulsions are made on ice (to prevent reaction) as inexample 1. The first emulsion is made by dispersing a mixture of 100 μMcompound 2 (FIG. 1), which has a bis-difluoromethylene phosphonate andis a known PTP1B inhibitor (Johnson et al., 2002), the target enzyme(human recombinant PTP1B, residues 1-322; Biomol Research Laboratories,Inc.) at 5 mU/ml, the fluorogenic PTP1B substrate6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular Probes),and a pre-defined ratio of Qdot™ Streptavidin Conjugates with emmissionmaxima at 585 nm, 655 nm and 705 nm (Quantum Dot Corporation, HaywardCalif.) in a buffer compatible with PTP1B activity (25 mM HEPES, pH 7.4,125 mM NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002). The 99 otherwater-in-oil emulsions are identical to the above but each contain oneof 99 carboxylic acids from the Carboxylic Acid Organic Building BlockLibrary (Aldrich) in place of compound 2, and different ratios of Qdot™Streptavidin Conjugates with emmission maxima at 585 nm, 655 nm and 705nm. In all emulsions the concentration of the 705 nm Qdot™ StreptavidinConjugates is 100 nM, and the concentrations of the 585 nm and 655 nmQdot™ Streptavidin Conjugates is either 0, 11, 22, 33, 44, 55, 66, 77,88 or 100 nM. Hence, there are 100 (10×10) permutations of Qdot™Streptavidin Conjugate concentrations which allows the microcapsulescontaining each compound to have a unique fluorescence signature whichis read by determining the fluorescence ratios of fluorescence at 705nm, 585 nm and 655 nm.

The 100 emulsions are mixed in equal ratios by vortexing and thetemperature raised to 25° C. for 30 min. Inhibitors reduce the amount ofnon-fluorescent substrate (DiFMUP) converted to the dephosphorylatedproduct (DiFMU; excitation/emmission maxima 358/452 nm; bluefluorescence). The water-in-oil emulsion is then converted into awater-in-oil-in water double emulsion and analysed by multi-colour flowcytometery as in example ln. Predominantly, all microcapsules exhibitedblue fluorescence due to dephosphorylation of DiFMUP by PTP1B exceptthose with the Qdot fluorescence signature of the microcapsulescontaining compound 2.

Example 4

Screening of PTP1B Inhibitors from a Compound Library

100 aqueous mixtures are made on ice (to prevent reaction). The firstmixture contains 100 μM compound 2 (FIG. 1), which has abis-difluoromethylene phosphonate and is a known PTP1B inhibitor(Johnson et al., 2002), the target enzyme (human recombinant PTP1B,residues 1-322; Biomol Research Laboratories, Inc.) at 5 mU/ml, thefluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferyl phosphate(DiFMUP) (Molecular Probes), and a pre-defined ratio of Qdot™Streptavidin Conjugates with emmission maxima at 585 nm, 655 nm and 705nm (Quantum Dot Corporation, Hayward Calif.) in a buffer compatible withPTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1 mMEDTA) (Doman et al., 2002). The 99 other aqueous mixtures are identicalto the above but each contain one of 99 carboxylic acids from theCarboxylic Acid Organic Building Block Library (Aldrich) in place ofcompound 2, and different ratios of Qdot™ Streptavidin Conjugates withemission maxima at 585 nm, 655 nm and 705 nm. In all mixtures theconcentration of the 705 nm Qdot™ Streptavidin Conjugates is 100 nM, andthe concentrations of the 585 nm and 655 nm Qdot™ StreptavidinConjugates is either 0, 11, 22, 33, 44, 55, 66, 77, 88 or 100 nM. Hence,there are 100 (10×10) permutations of Qdot™ Streptavidin Conjugateconcentrations which allows the microcapsules containing each compoundto have a unique fluorescence signature which is read by determining thefluorescence ratio of fluorescence at 705 nm, 585 nm and 655 nm.

0.5 μl of each of the compound mixtures is added sequentially to asolution of 1% (w/v) Span 60 and 1% (w/v) cholesterol in decane, madeand held at 37° C. as example 1, whilst homogenising at 25,000 r.p.m.using an Ultra-Turrax T8 Homogenizer (IKA) with a 5 mm dispersing tool.Homogenisation is continued for 3 minutes after the addition of thesecond aliquot. The coarse emulsion produced is then extruded as inexample 1 to create a fine water-in-oil emulsion and incubated at 37° C.for 30 min. Inhibitors reduce the amount of non-fluorescent substrate(DiFMUP) converted to the dephosphorylated product (DiFMU;excitation/emmission maxima 358/452 nm; blue fluorescence). Thewater-in-oil emulsion is then converted into a water-in-oil-in waterdouble emulsion and analysed by multi-colour flow cytometery as inexample 1. Predominantly, all microcapsules exhibited blue fluorescencedue to dephosphorylation of DiFMUP by PTP1B except those with the Qdotfluorescence signature of the microcapsules containing compound 2.

Example 5

Screening for PTP1B Inhibitors Using Microcapsules in MicrofluidicSystems

Microchannels are fabricated with rectangular cross-sections using rapidprototyping in poly(dimethylsiloxane) (PDMS) (McDonald and Whitesides,2002) and rendered hydrophobic as (Song and Ismagilov, 2003). Syringepumps were used to drive flows (Harvard Apparatus PHD 2000 Infusionpumps). For aqueous solutions, 50 μl Hamilton Gastight syringes (1700series, TLL) with removeable needles of 27-gaugeare used with 30-gaugeTeflon tubing (Weico Wire and Cable). For the carrier fluid, 1 mlHamilton Gastight syringes (1700 series, TLL) are used with 30-gaugeTeflon needles with one hub from Hamilton (Song and Ismagilov, 2003).The carrier fluid is 9% (v/v) C₆F₁₁C₂H₄OH in perfluorodecaline (PFD)(Song et al., 2003). All water-soluble reagents were dissolved in (25 mMHEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA), a buffer compatible with PTP1Bactivity.

A solution of the target enzyme (human recombinant PTP1B, residues1-322; Biomol Research Laboratories, Inc.) at 50 mU/ml and a solution ofeither a) 100 μM compound 2 (FIG. 1), which has a bis-difluoromethylenephosphonate and is a known PTP1B inhibitor (Johnson et al., 2002), or b)100 μM hydrocinnamic acid (Aldrich), a compound that is not a PTPinhibitor are flowed in a microchannel as two laminar streams, with aninert centre stream (of 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA) toseparate them and prevent the enzyme and compound coming into contactprior to droplet microcapsule formation (Song et al., 2003). These threesteams are continuously injected into a flow of water immisciblefluorocarbon carrier fluid (9% (v/v) C₆F₁₁C₂H₄OH in PFD). Inlet channelsfor the aqeous solutions are 50 μm² wide and channel for PFD is 28 μmwide. A variety of PFD/water volumetric flow rates (in μl min⁻¹) can beused including 0.6:0.3, 1.0:0.6, 12.3:3.7, 10:6 and 20:6, resulting inflow rates of 10, 19, 190, 190 and 300 mm s⁻¹ respectively. Aqueousmicrocapsules which occupy the entire width of the channel are formed bydrop-breakoff in the PFD stream (Song et al., 2003). Microcapsulescontaining either compound 2 or hydrocinnamic acid can be formed byswitching between injection with syringes containing compound 2 andhydrocinnamic acid.

The channel immediately downstream of the point of droplet formation iswinding with a peak to peak distance of 50 μm for a distance of 1 mm.This results in rapid mixing of the contents of the microcapsule bychaotic advection (Song et al., 2003). After this point themicrocapsules are run for up to 1 min through a 60 cm long microchannel(to allow inhibitor binding). This microchannel is then merged with a60×50 μm² microchannel containing aqueous microcapsules in (9% (v/v)C₆F₁₁C₂H₄OH in PFD) formed as above. These larger microcapsules containthe fluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP) (Molecular Probes) in 25 mM HEPES, pH 7.4, 125 mMNaCl, 1 mM EDTA. After the junction between the microchannels theexpanded main channel is 100×50 μm² and the microcapsules do not blockthe channel and can move at different speeds until a large microcapsule(containing DiFMUP) coalesces with a small microcapsule (containingPTP1B and the compound) (Song et al., 2003). The frequency of productionof large and small microcapsules is equal such that each largemicrocapsule has a small microcapsule with which to fuse. The fusedmicrocapsules are then run for up to 2 min through a 60 cm longmicrochannel. Fluorescence of the microcapsules due to production ofDiFMU (excitation/emmission maxima 350/452 nm; blue fluorescence) ismeasured using an epifluorescence microscope. Predominantly,microcapsules exhibiting blue fluorescence are those containinghydrocinnamic acid whereas microcapsules containing compound 2 exhibitlow fluorescence due to inhibition of PTP1B.

Example 6

Attachment of a Compound Library to Microbeads

5.5 μM diameter polystyrene microbeads that bear carboxylate functionalgroups on the surface are commercially available (www.luminexcorp.com)in an optically tagged form, as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997). A set of 100 such beads, each with a unique opticalsignature (www.luminexcorp.com) are modified with an excess ofethylenediamine and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (Pierce) as (Hermanson, 1996) to create primary aminogroups on the surface. The photocleavable linker4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid (NovaBiochem)(Holmes and Jones, 1995) is then attached to the beads by forming anamide bond using EDC as above. 100 different carboxylic acids from theCarboxylic Acid Organic Building Block Library (Aldrich) are thencoupled to the beads, by reacting with the linker alcohol to form acarboxylate ester, each of the 100 different optically tagged beadsbeing coupled to a different carboxylic acid, and each bead beingderivatised with ˜10⁶ molecules of carboxylic acid. Irradiation for 4min on ice using a B100 AP 354 nm UV lamp (UVP) from a distance of ˜5 cmresults in release of the compounds from the beads as carboxylic acids.

Example 7

Screening for Inhibitors of the Enzyme Protein Tyrosine Phosphatase 1B(PTP1B) Using Compounds Attached to Microbeads

PTP1B is a negative regulator of insulin and leptin signal transduction.Resistance to insulin and leptin are hallmarks of type 2 diabetesmellitus and obesity and hence PTP1B is an attractive drug target fordiabetes and obesity therapy (Johnson et al., 2002). 5.5 μm diameterpolystyrene microbeads that bear carboxylate functional groups on thesurface are commercially available (www.luminexcorp.com) in an opticallytagged form, as a result of incorporation of precise ratios of orange(585 nm), and red (>650 nm) fluorochromes (Fulton et al., 1997). First,the carboxylate functional groups on the microbeads are converted toprimary amines using ethylenediamine and EDC as in example 6. Aphosphopeptide substrate for PTP1B, the undecapaptide EGFR₉₈₈₋₉₉₈(DADEpYLIPQQG) (Zhang et al., 1993), is then coupled to both sets ofmicrobeads via the surface amino groups using EDC. This peptide is madeby solid phase synthesis on Sieber Amide resin(9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin) (Novabiochem) withorthogonal protection on the side chain carboxylate groups usingcarboxylate-O-allyl esters. A linker comprised of tetradecanedioic acidis coupled to the N-terminus and the peptide cleaved from the beadsusing 1% TFA to yield a peptide with a C-terminal amide The peptide iscoupled to the beads (using EDC) via the linker to give ˜10⁵ peptidesper bead. The remaining surface amino groups are then modified byattaching the photochemically cleavable linker4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid as in example6. The protecting groups on the side chain carboxylates of the peptideare then removed using Pd(Ph₃)₄/CHCl₃/HOAc/N-methyl morpholine. A firstset of microbeads is derivatised with3-(4-difluorophosphonomethylphenyl)propanoic acid (compound 1, FIG. 1),a compound that is a known PTP1B inhibitor (Johnson et al., 2002). Asecond set of beads, with a distinct optical tag from the first set ofbeads, is derivatised with hydrocinnamic acid (Aldrich), a compound thatis not a PTP1B inhibitor. In each case the compound is coupled byreacting with the linker alcohol to form a carboxylate ester as inexample 6. Each microbead is derivatised with ˜10⁶ molecules (Fulton etal., 1997).

The microbeads are then screened using the method outlined in FIG. 2.The two sets of microbeads are mixed in ratios varying from 1:1000 to1:1 (compound 1 beads: hydrocinnamic acid beads) and 10⁸ totalmicrobeads are mixed with the target enzyme (human recombinant PTP1B,residues 1-322; Biomol Research Laboratories, Inc.) at a concentrationof 10 nM, on ice (to prevent reaction) in a buffer compatible with PTP1Bactivity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA)(Doman et al., 2002). Single beads and target enzyme (PTP1B) are thencolocalised in microcapsules by forming a water-in-oil emulsion (also onice). The concentration of beads is such that most microcapsules containone or no beads. The compound is released photochemically (as in example6) and the temperature raised to 25° C. Inhibitors reduce the amount ofsubstrate converted to product (dephosphorylated peptide). The emulsionis cooled to 4° C. and broken as (Griffiths and Tawfik, 2003) into 100μl vanadate to stop the reaction (Harder et al., 1994). After labellingwith an anti-substrate (anti-phosphotyrosine) antibody labelled with thegreen (530 nm) fluorochrome fluorescein isothiocyanate (mouse monoclonalIgG_(2b) PY20 (Santa Cruz) according to the manufacturer's instructions,beads are analysed by 3-colour flow cytometry using a FACScan(Becton-Dickinson), FACScalibur (Becton-Dickinson) or MoFlo (Cytomation)flow cytometers to simultaneously determine the extent of inhibition andthe compound on the beads. Predominantly, dephosphorylation of thepeptide is only observed on those microbeads which were coated withPTP1B inhibitors, and not on other microbeads.

Example 8

Screening of PTP1B Inhibitors from a Compound Library Attached toMicrobeads

A set of 100 5.5 μM diameter polystyrene microbeads, bearing carboxylatefunctional groups on the surface and each with a unique opticalsignature (www.luminexcorp.com) as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997) are derivatised with a phosphopeptide substrate for PTP1B,the undecapaptide EGFR₉₈₈₋₉₉₈ (DADEpYLIPQQG) (Zhang et al., 1993), and100 different carboxylic acids, each attached via a photochemicallycleavable linker, as in example 7. One of these carboxylic acids is3-(4-difluorophosphonomethylphenyl)propanoic acid (compound 1, FIG. 1),a compound that is a known PTP1B inhibitor (Johnson et al., 2002). Theother 99 carboxylic acids are from the Carboxylic Acid Organic BuildingBlock Library (Aldrich) as example 6. Equal numbers of each of the 100bead sets are then mixed and screened as for example 7. Predominantly,dephosphorylation of the peptide is only observed on those microbeadswhich were coated with the PTP1B inhibitor3-(4-difluorophosphonomethylphenyl)propanoic acid (compound 1, FIG. 1),and not on microbeads coated with other compounds.

Example 9 Synthesis of Secondary Compounds in Emulsion Microcapsules andScreening for PTP1B Inhibition in Microfluidic System

Microchannels are fabricated with rectangular cross-sections using rapidprototyping in poly(dimethylsiloxane) (PDMS) (McDonald and Whitesides,2002) and rendered hydrophobic as (Song and Ismagilov, 2003). Syringepumps were used to drive flows (Harvard Apparatus PHD 2000 Infusionpumps). For aqueous solutions, 50 μl Hamilton Gastight syringes (1700series, TLL) with removeable needles of 27-gaugeare used with 30-gaugeTeflon tubing (Weico Wire and Cable). For the carrier fluid, 1 mlHamilton Gastight syringes (1700 series, TLL) are used with 30-gaugeTeflon needles with one hub from Hamilton (Song and Ismagilov, 2003).The carrier fluid is 9% (v/v) C₆F₁₁C₂H₄OH in perfluorodecaline (PFD)(Song et al., 2003). All water-soluble reagents were dissolved in (25 mMHEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA), a buffer compatible with PTPactivity.

A solution of the target enzyme (human recombinant PTP1B, residues1-322; Biomol Research Laboratories, Inc.) at 50 mU/ml, a solution of acompound which is a primary amine, and a solution of a compound which isan aldehyde are flowed in a microchannel as three laminar streams, withtwo inert separating streams (of 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mMEDTA) to prevent the enzyme and the compounds coming into contact priorto droplet microcapsule formation (Song et al., 2003). These fivestreams are continuously injected into a flow of water immisciblefluorocarbon carrier fluid (9% (v/v) C₆F₁₁C₂H₄OH in PFD). The amines andaldehydes can either a) contain a difluoromethylene phosphonate moiety(FIG. 6, compounds A and B), or b) contain no difluoromethylenephosphonate moiety.

Inlet channels for the aqeous solutions are 50 μm² wide and the channelfor PFD is 28 μm wide. A variety of PFD/water volumetric flow rates (inμl min⁻¹) can be used including 0.6:0.3, 1.0:0.6, 12.3:3.7, 10:6 and20:6, resulting in flow rates of 10, 19, 190, 190 and 300 mm s⁻¹respectively. Aqueous microcapsules which occupy the entire width of thechannel are formed by drop-breakoff in the PFD stream (Song et al.,2003). Microcapsules containing compounds with and withoutdifluoromethylene phosphonate moieties can be formed by switchingbetween injection with syringes containing amines or aldehydes with orwithout a difluoromethylene phosphonate moiety.

The channel immediately downstream of the point of droplet formation iswinding with a peak to peak distance of 50 μM for a distance of 1 mm.This results in rapid mixing of the contents of the microcapsule bychaotic advection (Song et al., 2003). After this point themicrocapsules are run for up to 1 min through a 60 cm long microchannel.This allows the amine and the aldehyde to react together by formation ofa Schiff base to create a secondary compound and allows inhibitors tobind to PTP1B. This microchannel is then merged with a 60×50 μm²microchannel containing aqueous microcapsules in (9% (v/v) C₆F₁₁C₂H₄OHin PFD) formed as above. These larger microcapsules contain thefluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferyl phosphate(DiFMUP) (Molecular Probes) in 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mMEDTA. After the junction between the microchannels the expanded mainchannel is 100×50 μm² and the microcapsules do not block the channel andcan move at different speeds until a large microcapsule (containingDiFMUP) coalesces with a small microcapsule (containing PTP1B and thecompound) (Song et al., 2003). The frequency of production of large andsmall microcapsules is equal such that each large microcapsule has asmall microcapsule with which to fuse. The fused microcapsules are thenrun for up to 2 min through a 60 cm long microchannel. Fluorescence ofthe microcapsules due to production of DiFMU (excitation/emmissionmaxima 358/452 nm; blue fluorescence) is measured using anepifluorescence microscope.

Predominantly, when the amine and aldehyde concentrations are low (<100μM) inhibition of PTP1B activity is only observed in microcapsulescontaining both an amine with a difluoromethylene phosphonate moiety(compound A, FIG. 6) and an aldehyde with a difluoromethylenephosphonate moiety (compound B, FIG. 6). This is because the Schiff baseformed in these microcapsules (compound C, FIG. 6) containsbis-difluoromethylene phosphonate and is a much more potent PTP1Binhibitor than a molecule with a single difluoromethylene phosphonatemoiety (see FIG. 1).

Predominantly, when the amine and aldehyde concentrations are high (>100μM) inhibition of PTP1B activity is observed in microcapsules containingeither an amine with a difluoromethylene phosphonate moiety (compound A,FIG. 6) or an aldehyde with a difluoromethylene phosphonate moiety(compound B, FIG. 6), or both, but not in other microcapsules. This isbecause at higher concentrations molecules with either a singledifluoromethylene phosphonate moiety or a bis-difluoromethylenephosphonate (compound C, FIG. 6) can inhibit PTP (see FIG. 1).

Example 10

Synthesis of Secondary Compounds in Emulsion Microcapsules and Screeningfor PTP1B Inhibition Using Compounds Attached to Microbeads

5.5 μm diameter polystyrene microbeads that bear carboxylate functionalgroups on the surface are commercially available (www.luminexcorp.com)in an optically tagged form, as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997). First, the carboxylate functional groups on the microbeadsare converted to primary amines using ethylenediamine and EDC as inexample 6. A phosphopeptide substrate for PTP1B, the undecapaptideEGFR₉₈₈₋₉₉₈ (DADEpYLIPQQG) (Zhang et al., 1993), is then coupled to bothsets of microbeads via the surface amino groups using EDC and theprotecting groups on the side chain carboxylates of the peptide removedas in example 7. A first set of microbeads (set 1) is reacted withsuccinimidyl p-formylbenzoate to convert the surface amino groups toaldehydes. A second set of microbeads (set 2), with a distinct opticaltag from the first set of microbeads, is left unreacted (i.e. withprimary amines on the surface).

The first set of microbeads (set 1), are then reacted with a compoundcontaining a difluoromethylene phosphonate moiety and a primary amine(compound A, FIG. 6) via reaction with the surface aldehyde groups toform a Schiff base. The second set of microbeads (set 2), with adistinct optical tag from the first set of beads, are reacted with acompound containing a difluoromethylene phosphonate moiety and analdehyde (compound B, FIG. 6) via reaction of the aldehyde with thesurface amine groups to form a Schiff base. The formation of Schiffbases is enhanced by reaction at alkaline pH (i.e. pH9-10). Microbeadscoated with compounds at various densities are created.

The two sets of microbeads are mixed with the target enzyme (humanrecombinant PTP1B, residues 1-322; Biomol Research Laboratories, Inc.)at a concentration of 10 nM, on ice (to prevent reaction) in a buffercompatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10%glycerol, 1 mM EDTA) (Doman et al., 2002). The beads and target enzyme(PTP1B) are immediately compartmentalised in microcapsules by forming awater-in-oil emulsion (also on ice).

The number of microbeads is varied such that, at one extreme, mostmicrocapsules contain one or no beads, and at the other, the majority ofmicrocapsules contain two or more microbeads. The temperature raised to25° C. The Schiff base is a relatively labile, reversible interaction,readily hydrolysed at neutral pH, resulting in release of compounds fromthe beads. In microcapsules containing a microbead from both set 1 andset 2, the compounds released from the microbeads can react with eachother, forming a Schiff base and creating a new molecule in solution.This new molecule (FIG. 6, compound C) contains a bis-difluoromethylenephosphonate moiety and has significantly more potency as a PTP1Binhibitor than compounds with a single difluoromethylene phosphonatemoiety (see FIG. 1). Inhibitors reduce the amount of substrate convertedto product (dephosphorylated peptide). The emulsion is cooled to 4° C.and broken as (Griffiths and Tawfik, 2003) into 100 μM vanadate to stopthe reaction (Harder et al., 1994). After labelling with ananti-substrate (anti-phosphotyrosine) antibody labelled with the green(530 nm) fluorochrome fluorescein isothiocyanate (mouse monoclonal IgG₂,PY20 (Santa Cruz) according to the manufacturer's instructions, beadsare analysed by 3-colour flow cytometry using a FACScan(Becton-Dickinson), FACScalibur (Becton-Dickinson) or MoFlo (Cytomation)flow cytometers to simultaneously determine the extent of inhibition andthe compound on the beads. With low microbead numbers, mostmicrocapsules contain only a single or no microbeads and PTP1Binhibition is only detected on beads coated with a high density ofinhibitor, when the concentration of inhibitor released into solution ineach microcapsule is sufficiently high for effective inhibition. Incontrast, when the bead numbers are higher, many microbeads are detectedwhere little substrate has been converted to product, even when themicrobeads are coated with inhibitor at low density. This is due to theformation of a highly potent PTP1B inhibitor (FIG. 6, compound C)containing a bis-difluoromethylene phosphonate moiety in microcapsulescontaining a microbead each from set 1 and set 2.

Example 11

Synthesis of a Library 2500 Secondary Compounds in EmulsionMicrocapsules and Screening for PTP1B Inhibition Using CompoundsAttached to Microbeads

A set of 100 5.5 μm diameter polystyrene microbeads, bearing carboxylatefunctional groups on the surface and each with a unique opticalsignature (www.luminexcorp.com) as a result of incorporation of preciseratios of orange (585 nm), and red (>650 nm) fluorochromes (Fulton etal., 1997) are modified to convert the carboxylate functional groups toprimary amines as in example 6, then derivatised with a phosphopeptidesubstrate for PTP1B, the undecapaptide EGFR₉₈₈₋₉₉₈ (DADEpYLIPQQG) (Zhanget al., 1993), as in example 10. The first 50 sets of microbeads arereacted to convert a proportion of the surface carboxyl groups toaldehydes as in example 10. The second 50 sets of microbeads are leftunreacted (i.e. with primary amines on the surface).

The first 50 sets of microbeads are each reacted with a unique compoundcontaining a primary amine via reaction with the surface aldehyde groupsto form a Schiff base which links the compounds to the beads. One ofthese compounds (compound A, FIG. 6) contains a difluoromethylenephosphonate moiety. The second 50 sets of microbeads are each reactedwith a unique compound containing an aldehyde via reaction with thesurface amine groups to form a Schiff base which links the compounds tothe beads. One of these compounds (compound B, FIG. 6) contains adifluoromethylene phosphonate moiety. The formation of Schiff bases isenhanced by reaction at alkaline pH (i.e. pH9-10).

The two sets of microbeads are mixed with the target enzyme (humanrecombinant PTP1B, residues 1-322; Biomol Research Laboratories, Inc.)at a concentration of 10 nM, on ice (to prevent reaction) in a buffercompatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10%glycerol, 1 mM EDTA) (Doman et al., 2002). The beads and target enzyme(PTP 1 B) are immediately compartmentalised in microcapsules by forminga water-in-oil emulsion (also on ice).

The number of microbeads is such that the modal number of microcapsulesper microcapsule is two. The temperature raised to 25° C. The Schiffbase is a relatively labile, reversible interaction, readily hydrolysedat neutral pH, resulting in release of compounds from the beads. Inmicrocapsules containing a microbead from one of the first 50 sets and amicrobead from one of the second 50 sets, the compounds released fromthe microbeads can react with each other, forming a Schiff base andcreating a new molecule in solution. Inhibitors reduce the amount ofsubstrate converted to product (dephosphorylated peptide). The emulsionis cooled to 4° C. and broken as (Griffiths and Tawfik, 2003) into 100μM vanadate to stop the reaction (Harder et al., 1994). After labellingwith an anti-substrate (anti-phosphotyrosine) antibody labelled with thegreen (530 nm) fluorochrome fluorescein isothiocyanate (mouse monoclonalIgG_(2b) PY20 (Santa Cruz) according to the manufacturer's instructions,beads are analysed by 3-colour flow cytometry using a FACScan(Becton-Dickinson), FACScalibur (Becton-Dickinson) or MoFlo (Cytomation)flow cytometers to simultaneously determine the extent of inhibition andthe compound on the beads. Beads which were coated with a primarycompound which is itself a PTP1B inhibitor, or which reacts with anotherprimary compound released from another co-compartmentalised bead to forma second inhibitor are identified as little substrate has been convertedto product. The identified beads where as little substrate has beenconverted to product include those carrying compounds containing adifluoromethylene phosphonate moiety. When the microbeads are coatedwith compounds at low density the concentration of the releasedcompounds containing a difluoromethylene phosphonate moieties in themicrocapsules is insufficient to efficiently inhibit PTP1B (see example7). However in microcapsules containing two microbeads, one from thefirst set of 50 beads and one from the second set of 50 beads, and whereeach microbead carries a molecule with a difluoromethylene phosphonatemoiety, the released molecules can form a highly potent PTP inhibitor(compound C, FIG. 6) containing a bis-difluoromethylene phosphonatemoiety in the microcapsules which inhibits conversion of the PTP1Bsubstrate to product.

Example 12

Compartmentalisation of Small Molecules in a Water-in-FluorocarbonEmulsions.

Water-in-fluorocarbon emulsions containing 95% (v/v) perfluorooctylbromide, 5% (v/v) phosphate buffered saline containing the molecule ofinterest in solution, and 2% (w/v) C₈F₁₇C₁₁H₂₂OP(O)[N(CH₂CH₂)₂O]₂(F8H11DMP) as surfactant were formed essentially as (Sadtler et al.,1996) by extrusion (15 times) through 14 μm filters (Osmonics) or byhomogenising for 5 mm at 25,000 r.p.m. using an Ultra-Turrax T8Homogenizer (IKA) with a 5 mm dispersing tool. Emulsions were madecontaining a series of small fluorescent molecules dissolved in theaqueous phase at concentrations from 100 μm to 2 mM. These molecules,including calcein, texas red, fluorescein, coumarin 102,7-hydroxycoumarin-3-carboxylic acid and 7-diethylamino-4-methyl coumarin(coumarin 1), had molecular weights from 203 to 625 Da and LogPvalues—calculated using SRC's LogKow/KowWin Program (Meylan and Howard,1995)—ranging from −0.49 to 4.09. Emulsions containing differentcoloured fluorochromes were mixed by vortexing. Compartmentalisation wasobserved by epifluorescence microscopy of the mixed emulsions. Noexchange between compartments was observed 24 hours after mixing (seeFIG. 5).

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All publications mentioned in the above specification, and referencescited in said publications, are herein incorporated by reference.Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. A method of forming a small molecule compound comprising: providingat least a first aqueous fluid comprising at least a first smallmolecule compound having a molecular weight of less than 500 Daltons;providing at least a second aqueous fluid comprising at least a secondsmall molecule compound having a molecular weight of less than 500Daltons; encapsulating said at least first small molecule compound intoat least a first aqueous microcapsule within an immiscible fluorocarbonoil; encapsulating said at least second small molecule compound into atleast a second aqueous microcapsule within an immiscible fluorocarbonoil; and fusing said at least first and said at least second aqueousmicrocapsules, thereby causing a chemical reaction between said at leastfirst and at least second small molecule compounds to form at least athird small molecule compound.
 2. The method of claim 1, wherein said atleast first small molecule compound is attached to a microbead.
 3. Themethod of claim 1, wherein said at least second small molecule compoundis attached to a microbead.
 4. The method of claim 1, further comprisingisolating said at least third small molecule compound.
 5. The method ofclaim 1, further comprising identifying said at least third smallmolecule compound.
 6. The method of claim 5, wherein said at least thirdsmall molecule compound is identified by its optical properties.
 7. Themethod of claim 1, further comprising identifying said at least firstand at least second small molecule compounds which reacted to form saidat least third small molecule compound.
 8. The method of claim 1,wherein said at least first small molecule compound comprises at least afirst label and said at least second small molecule compound comprisesat least a second label, wherein said at least first and said at leastsecond labels are different.
 9. The method of claim 1, wherein said atleast first aqueous microcapsule comprises no more than one said atleast first small molecule compound.
 10. The method of claim 1, whereinsaid at least first aqueous microcapsule comprises a plurality of saidat least first small molecule compounds.
 11. The method of claim 1,wherein said at least second aqueous microcapsule comprises no more thanone said at least second small molecule compound.
 12. The method ofclaim 1, wherein said at least second aqueous microcapsule comprises aplurality of said at least second small molecule compounds.